Selective grp94 inhibitors and uses thereof

ABSTRACT

The disclosure relates to novel selective Grp94 inhibitors, compositions comprising an effective amount of such compounds, and methods to treat or prevent a condition, such as cancer, comprising administering to an animal in need thereof an effective amount of such compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/912,082, filed Feb. 12, 2016, which is the national phase ofInternational Patent Application No. PCT/US2014/051332, filed Aug. 15,2014, which claims priority to U.S. provisional patent application No.61/866,932, filed Aug. 16, 2013, the entire contents of which are herebyincorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under AI090501 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 12, 2014, isnamed 2003080-0708_SL.txt and is 17,757 bytes in size.

FIELD

The disclosure relates to selective Grp94 inhibitors, compositionscomprising an effective amount of such compounds, and methods to treator prevent a condition, such as cancer, comprising administering to ananimal in need thereof an effective amount of such compounds.

BACKGROUND

The Hsp90s are a family of molecular chaperones that play importantroles in regulating and maintaining the functionality of cells underproteotoxic stress and pathogenic pressure (Workman, P., Burrows, F.,Neckers, L. & Rosen, N. Drugging the cancer chaperone Hsp90:combinatorial therapeutic exploitation of oncogene addiction and tumorstress. Ann. N.Y. Acad. Sci. 1113, 202-216 (2007)). In humans,cytoplasmic heat shock protein 90 alpha and beta (Hsp90α and β),endoplasmic reticulum (ER) glucose-regulated protein 94 (Grp94) and themitochondrial tumor necrosis factor receptor-associated protein 1(Trap-1) are the four known Hsp90 paralogs (Sreedhar, A. S., Kalmar, E.,Csermely, P. & Shen, Y. F. Hsp90 isoforms: functions, expression andclinical importance. FEBS letters. 562, 11-15 (2004); Johnson, J. L.Evolution and function of diverse Hsp90 homologs and cochaperoneproteins. Biochim. Biophys. Acta. 1823, 607-613 (2012)). These proteinsare ATP dependent and belong to the GHKL (Gyrase B, Hsp90, HistidineKinases, MutL) ATPase superfamily, which is characterized by a distinctATP binding “Bergerat fold” located in the N-terminal domain (NTD)(Chene, P. ATPases as drug targets: learning from their structure. Nat.Rev. Drug Discov. 1, 665-673 (2002)). Binding and release of thenucleotide drives the catalytic cycle of the Hsp90s and thereby assistsin the refolding of client proteins through a series ofassociation-dissociation catalytic cycles. Occupancy of this regulatorypocket by small molecule inhibitors inactivates Hsp90 chaperonefunction, and several pan-Hsp90 inhibitors have demonstrated potentreversal of the disease phenotype when tested in models of cancer,neurodegeneration, infection, and inflammatory disease. Due to thesetherapeutic activities, a select number of these compounds have alsomoved to the clinic for the treatment of cancers (Jhaveri, K., Taldone,T., Modi, S. & Chiosis, G. Advances in the clinical development of heatshock protein 90 (Hsp90) inhibitors in cancers. Biochim. Biophys. Acta.1823, 742-755 (2012))

Despite considerable interest in the use of pharmacologic Hsp90inhibitors for the treatment of disease, little is known about thecontribution of each paralog to the observed therapeutic benefit. Todate, all published studies have used pan-Hsp90 inhibitors to inactivateHsp90s and the processes that depend on them, making it impossible tocorrelate the role of individual paralogs with the biological effects.This is particularly unsatisfying, considering that the chaperoningroles of these Hsp90s do not overlap. Thus, for example, while there isa considerable literature on the response of cytosolic Hsp90 toinhibitors, no study satisfactorily differentiates the role of the a and3 paralogs. Furthermore, although both Grp94 and Trap-1 are abundant inthe cancer cell, little is known about their contribution to themalignant phenotype (Sreedhar, A. S., Kalmar, E., Csermely, P. & Shen,Y. F. Hsp90 isoforms: functions, expression and clinical importance.FEBS letters. 562, 11-15 (2004); Johnson, J. L. Evolution and functionof diverse Hsp90 homologs and cochaperone proteins. Biochim. Biophys.Acta. 1823, 607-613 (2012); Marzec, M, Eletto, D. & Argon, Y. GRP94: AnHSP90-like protein specialized for protein folding and quality controlin the endoplasmic reticulum. Biochim. Biophys. Acta. 1823, 774-787(2012); Chen, B. The HSP90 family of genes in the human genome: Insightsinto their divergence and evolution. Genomics 86, 627-637 (2005)).

In large part the predicament of being unable to study individualparalogs in cancer cells, despite their divergent roles, stems from alack of suitable tools. While pan-Hsp90 inhibitors, geneticmanipulations in yeast and human cells, mutant cell lines, and genedeficient mice have shed light on several Hsp90-dependent cancermechanisms, many challenges still remain. In particular, strategies thataddress the biology of Hsp90s and their individual paralogs in anendogenous cellular environment where the chaperones are limiting butnot absent (i.e. in un-engineered cancer cell lines and in primarysamples) are needed. Ideally, this gap can be filled by chemical toolsthat probe and manipulate a protein's function in a controlled manner.Such tools would complement traditional biochemical and biologicalapproaches by aiding the molecular characterization of biomolecules bothin vitro and within their natural biological contexts.

While useful both as therapeutics and as tools to dissect thecell-specific effects and mechanisms associated with Hsp90 paralogs inselect phenotypes, the discovery of paralog specific Hsp90 inhibitors isparticularly challenging because of a high degree of conservation intheir ATP regulatory ligand binding cavities, the pocket to which theknown synthetic ligands bind. Indeed, we and others found that mostreported Hsp90 inhibitors bind equally well to the majority of theseparalogs (Marzec, M., Eletto, D. & Argon, Y. GRP94: An HSP90-likeprotein specialized for protein folding and quality control in theendoplasmic reticulum. Biochim. Biophys. Acta. 1823, 774-787 (2012);Schulte, T. W. et al. Interaction of radicicol with members of the heatshock protein 90 family of molecular chaperones. Mol. Endo. 13,1435-1448 (1999). Crystal structures of the cytoplasmic Hsp90 (α and β)N-terminal domain, either in the apo form or in complex with regulatorynucleotides or small molecules, are essentially superimposable(Immormino, R. M., Kang, Y., Chiosis, G. & Gewirth, D. T. Structural andquantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90inhibitors. J. Med. Chem. 49, 4953-4960 (2006); Soldano, K. L., Jivan,A., Nicchitta, C. V. & Gewirth, D. T. Structure of the N-terminal domainof GRP94: Basis for ligand specificity and regulation. J. Biol. Chem.279, 48330-48338 (2003)). In addition, while slightly different dockingorientations were observed for some small molecule ligands when bound toHsp90 and Grp94, these have, as of yet, failed to translate intoappreciable selectivity and specific cellular activity throughindividual paralog inhibition (Marzec, M., Eletto, D. & Argon, Y. GRP94:An HSP90-like protein specialized for protein folding and qualitycontrol in the endoplasmic reticulum. Biochim. Biophys. Acta. 1823,774-787 (2012); Immormino, R. M. et al. Different poses for ligand andchaperone in inhibitor-bound Hsp90 and GRP94: implications forparalog-specific drug design. J. Mol. Biol. 388, 10331042 (2009);(Duerfeldt, A. S., et al. Development of a Grp94 inhibitor. J. Am. Chem.Soc. 134, 9796-9804 (2012)).

Paradoxically, despite the high degree of sequence conservation in theirATP binding pockets, crystallographic and biochemical studies have shownthat when bound to nucleotides, Hsp90α/β, Grp94 and Trap-1 adoptdistinctly different conformations and hydrolyze ATP with notablydifferent rates. Specifically, when bound to adenyl imidodiphosphate(AMP-PNP) a non-hydrolyzable ATP analog, the “lids” of the twoN-terminal domains (NTD) of the yeast Hsp90a dimer move from the “open”to the “closed” conformation, trapping the bound nucleotide within theATP binding cavity. The two closed NTDs then meet to form a second dimerinterface that supplements the obligatory dimeric interactionscontributed by the two C-terminal domains and importantly, aligns thecatalytic residues for ATP hydrolysis. In contrast, the NTD “lids” ofGrp94 do not close upon nucleotide binding but instead adopt a unique“extended open” conformation that does not cover the ATP binding pocketand does not allow for strong dimeric interactions between NTDs. As aresult, nucleotide-bound Grp94 adopts a twisted “V” shape with theirNTDs not symmetrically opposed, but, rather, oriented in oppositedirections (Ali, M. M. et al. Crystal structure of anHsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 440,1013-1017 (2006); Dollins, D. E., Immormino, R. M. & Gewirth, D. T.Structure of unliganded GRP94, the endoplasmic reticulum Hsp90. Basisfor nucleotide-induced conformational change. J. Biol. Chem. 280,30438-30447 (2005)). In Trap-1, ATP binding leads to a predominantlyclosed conformation, albeit with kinetics slower than in the cytosolicHsp90 (Leskovar, A., Wegele, H., Werbeck, N. D., Buchner, J. &Reinstein, J. The ATPase cycle of the mitochondrial Hsp90 analog Trap1.J. Biol. Chem. 283, 11677-11688 (2008)). Nonetheless this isinsufficient to commit Trap-1 to nucleotide hydrolysis and is insteadfollowed by re-opening of the chaperone conformation. Together, thebiochemical evidence suggests that the overall structure andconformational flexibility of the proteins plays an important role inconfiguring the ATP-binding sites of these chaperones.

In the present disclosure, we take advantage of the conformationaldistinctions between the paralogs and use the chemical diversityimprinted into the purine-scaffold class to demonstrate that theidentification of Hsp90 paralog-specific ligands is possible. We explainthe source of paralog binding specificity using structural and modelinganalyses. We then use several of the identified paralog specificinhibitors to provide novel insights into the tumor-specific chaperoningof a client protein by individual Hsp90s.

SUMMARY OF INVENTION

The present disclosure relates to the discovery that paralogs of Hsp90,although very similar, interact with structurally related inhibitors ina very different manner. An understanding of the structural attributesof these inhibitors and their binding to the target proteins has led tothe development of inhibitors that are selective for particular paralogsof Hsp90, as described herein. In particular, new compounds that showhigh specificity for Grp94 have been developed. In some embodiments,Grp94 selective compounds are capable of inhibiting Grp94 withoutinhibiting the other Hsp90 paralogs, including Hsp90α, Hsp90β andTrap-1. As a result, the selective Grp94 inhibitors of the disclosurecan be used in the treatment of various types of cancer. Moreover, thetherapeutic benefits can be obtained without a feed-back up-regulationof anti-apoptotic and resistance-mediating heat shock proteins, such asHsp70.

The disclosure provides evidence that Grp94 has an allosteric bindingsite that partially overlaps with the ATP/ADP binding site and containsa hydrophobic pocket that is not fully exposed in the other Hsp90paralogs. Grp94 inhibitors of the disclosure contain chemical moietiesthat can occupy the allosteric binding site and thus prevent binding ofATP/ADP.

The full length amino acid sequence of human Grp94 (SEQ ID NO: 1) isshown in Table 1. As discussed herein, selective Grp94 inhibitors of thedisclosure interact with specific amino acids comprising the N-terminaldomain (NTD) of Grp94. In particular, Grp94 inhibitors of the disclosurecan interact (e.g., make steric and electrostatic contacts) with twospecific binding sites of SEQ ID NO: 1, referred to herein as “bindingsite 1” and “binding site 2”. Binding site 1 is comprised of at leastfive amino acids which include Ile247, Val211, Phe199, Met154 andLeu163. Binding site 1 can also include the amino acids Leu159, Tyr200,and Trp223. Interaction of ligands (e.g., ATP or small moleculeinhibitors) with the amino acids comprising binding site 1 are conservedin all four paralogs—Grp94, Hsp90π, Hsp90β, and Trap-1. Binding site 2is comprised of at least seven amino acids of SEQ ID NO: 1, whichincludes Phe195, Gly198, Val209, Ala202, Leu104, Leu249 and Phe203.Binding site 2 (also referred to herein as the “Grp94 specific bindingsite”), which is specific for the Grp94 paralog, is located in the cleftregion adjoining the ATP/ADP binding site. Notably, access to bindingsite 2 is blocked by Phe138 in Hsp90a and Hsp90β, and Phe205 in Trap-1.Hence, Grp94 inhibitors of the disclosure are capable of interactingwith specific amino acids occupying binding site 2 of the Grp94 NTD,which allows for selective binding to the Grp94 paralog. In someembodiments, Grp94 inhibitors of the disclosure exhibit weaker bindingto the other Hsp90 paralogs than Grp94.

Accordingly, in one aspect, the disclosure provides new compounds thatexhibit affinity for Grp94 and thus, are capable of inhibiting thebiological activity of Grp94. In some embodiments, the Grp94 inhibitorsinteract with six or more of the amino acids comprising binding site 1and binding site 2 of the Grp94 NTD. In particular embodiments, theGrp94 inhibitors of the disclosure can interact with six, seven, eight,nine, ten, eleven or twelve of the amino acids comprising binding site 1and binding site 2 of the Grp94 NTD. In other embodiments, the Grp94inhibitors of the disclosure interact with six or more amino acidsselected from Phe195, Gly198, Val209, Ala202, Ile247, Leu249, Phe203,Leu104, Val211, Phe199, Met154 and Leu163 of SEQ ID NO:1. For instance,the Grp94 inhibitors of the disclosure can interact with six, seven,eight, nine, ten, eleven or twelve of the amino acids selected fromPhe195, Gly198, Val209, Ala202, Ile247, Leu249, Phe203, Leu104, Val211,Phe199, Met154 and Leu163 of SEQ ID NO. 1.

In particular embodiments, the Grp94 inhibitors of the disclosure arecapable of interacting with three or more of the amino acids in bindingsite 2 (i.e., the Grp94 selective binding site) of the Grp94 NTD. Forinstance, the Grp94 inhibitors can interact with three, four, five, sixor seven of the amino acids of binding site 2 of the Grp94 NTD. In somesuch embodiments, the Grp94 inhibitors of the disclosure are capable ofinteracting with three or more amino acids selected from Phe195, Gly198,Val209, Ala202, Leu104, Leu249 and Phe203 of SEQ ID NO: 1. For instance,the Grp94 inhibitors of the disclosure can interact with three, four,five, six or seven amino acids selected from Phe195, Gly198, Val209,Ala202, Leu104, Leu249 and Phe203 of SEQ ID NO:1.

In particular embodiments, the Grp94 selective inhibitors of thedisclosure are capable of interacting with the amino acids Ala202,Leu104 and Leu249 of SEQ ID NO: 1. In other embodiments, the Grp94selective inhibitors of the disclosure are capable of interacting withthe amino acids Gly198, Val209, Ala202, Leu249 and Phe203 of SEQ IDNO: 1. In other embodiments, the Grp94 selective inhibitors of thedisclosure are capable of interacting with the amino acids Phe195,Val209, Ala202 of SEQ ID NO: 1. In other embodiments, the Grp94selective inhibitors of the disclosure are capable of interacting withthe amino acids Leu104, Val209, Ala202 of SEQ ID NO: 1. In still otherembodiments, the Grp94 selective inhibitors of the disclosure arecapable of interacting with the amino acids Phe195, Leu249 and Leu104 ofSEQ ID NO: 1. In still other embodiments, the Grp94 selective inhibitorsof the disclosure are capable of interacting with the amino acidsPhe195, Gly198 and Val209 of SEQ ID NO:1. In still other embodiments,the Grp94 selective inhibitors of the disclosure are capable ofinteracting with the amino acids Leu104, Leu249 and Phe203 of SEQ ID NO:1.

The Grp94 inhibitors of the disclosure can be purine-scaffold compoundsor can be based on scaffolds related to purine (e.g., fused aminopyridine compounds). All Grp94 inhibitors that contain a purine scaffoldor a scaffold related to purine will be referred to hereinafter as apurine-scaffold inhibitor or a purine-scaffold compound. In someembodiments, the Grp94 inhibitors are adenine scaffold inhibitors. Insome embodiments, the Grp94 inhibitors are adenine scaffold compounds.

In particular embodiments, the purine-scaffold (e.g., adenine-scaffold)inhibitors can be substituted at the 8-position with a linker groupbonded to an aryl or heteroaryl group. For instance, the substituentbonded to the 8-position of the purine ring can be an arylsulfanylgroup, an arylsulfoxyl group, an arylsulfonyl group, a benzyl group, anarylcarbonyl group, an aniline group or a phenoxy group. In some suchembodiments, the aryl or heteroaryl group at the 8-position of thepurine ring interacts with amino acids comprising binding site 1 andbinding site 2 of SEQ ID NO: 1. For instance, the aryl or heteroarylgroup at the 8-position of the purine ring can interact with six, seven,eight, nine, ten, eleven or twelve of the amino acids selected fromPhe195, Gly198, Val209, Ala202, Ile247, Leu249, Phe203, Leu104, Val211,Phe199, Met154 and Leu163 of SEQ ID NO. 1. In other embodiments, thearyl or heteroaryl group at the 8-position of the purine ring caninteract with three, four, five, six or seven amino acids selected fromPhe195, Gly198, Val209, Ala202, Leu104, Leu249 and Phe203 of SEQ IDNO: 1. The purine portion of the purine-scaffold Grp94 inhibitors of thedisclosure generally interacts with amino acids that are conserved inall Hsp90 paralogs. For instance, the purine portion can form favorableinteractions with Asp149, Thr245, Ala111, Gly153, Lys114, Asp110, Ala108and Asn107 of SEQ ID NO:1.

In some embodiments, the Grp94 inhibitors of the disclosure are watersoluble. As used herein, water soluble is defined as having a solubilityof above 0.5 mg/mL in distilled water at ambient temperatures. In someembodiments, the water solubility of the purine-scaffold inhibitors ofthe disclosure can be greater than 3 mg/mL, greater than 4 mg/mL,greater than 5 mg/mL, greater than 10 mg/mL, greater than 20 mg/mL, orgreater than 40 mg/mL in distilled water at ambient temperatures. Aswill be discussed herein, the purine-scaffold inhibitors of thedisclosure can be formulated as salts to increase their watersolubility.

In one embodiment, the Grp94 inhibitor of the disclosure is a compoundof Formula (I). In another embodiment, the Grp94 inhibitor of thedisclosure is a compound of Formula (II). In another embodiment, theGrp94 inhibitor of the disclosure is a compound of Formula (III). Inanother embodiment, the Grp94 inhibitor of the disclosure is a compoundof Formula (IV). In another embodiment, the Grp94 inhibitor of thedisclosure is a compound of Formula (V).

Grp94 inhibitors of the disclosure are highly selective for Grp94relative to the other Hsp90 paralogs. In some embodiments, the Grp94inhibitors exhibit a greater than 10-fold preference for Grp94 overHsp90α, Hsp90β and/or Trap-1. In other embodiments, the Grp94 inhibitorsexhibit a greater than 20-fold preference for Grp94 over Hsp90α, Hsp90βand/or Trap-1. In yet other embodiments, the Grp94 inhibitors exhibit agreater than 50-fold preference for Grp94 over Hsp90α, Hsp90β and/orTrap-1. In yet other embodiments, the Grp94 inhibitors exhibit a greaterthan 100-fold preference for Grp94 over Hsp90α, Hsp90β and/or Trap-1. Inyet other embodiments, the Grp94 inhibitors exhibit a greater than500-fold preference for Grp94 over Hsp90α, Hsp90β and/or Trap-1. In someembodiments, the selectivity of the Grp94 inhibitors for binding toGrp94 over the other Hsp90 paralogs is measured using a fluorescencepolarization assay. For example, the selectivity may be measured in afluorescence polarization assay as described herein.

Grp94 inhibitors can be used to treat a variety of Hsp90 cancersincluding but not limited to colorectal cancer, pancreatic cancer,thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma,bladder cancer, prostate cancer, a lung cancer including small cell lungcancer and non-small cell lung cancer, breast cancer, neuroblastoma,gastrointestinal cancers including gastrointestinal stromal tumors,esophageal cancer, stomach cancer, liver cancer, gallbladder cancer,anal cancer, brain tumors including gliomas, lymphomas includingfollicular lymphoma and diffuse large B-cell lymphoma, leukemias,myelomas (e.g., multiple myeloma), myeloproliferative neoplasms andgynecologic cancers including ovarian, cervical, and endometrialcancers. In some embodiments, the Grp94 inhibitor can be used incombination with radiation therapy. In other embodiments, the Grp94inhibitor can be used in combination with a fluoropyrimidine-based orplatinum-based chemotherapy.

In particular embodiments, the Grp94 inhibitors of the disclosure can beused to treat human epidermal growth factor receptor 2 (HER2) dependentcancers such as breast cancer, ovarian cancer, gastric cancer,esophageal cancer and non-small-cell lung cancers. In some suchembodiments, the Grp94 inhibitors of the disclosure can be used incombination with a therapeutic reagent that interferes with the HER2receptor (e.g., trastuzumab (herceptin)).

In some embodiments, the Grp94 inhibitors of the disclosure can be usedto treat epidermal growth factor receptor (EGFR) dependent cancers suchas pancreatic cancer, neck cancer, breast cancer, ovarian cancer,cervical cancer, bladder and esophageal cancers. In some suchembodiments, the Grp94 inhibitors of the disclosure can be used to treatendocrine-resistant breast and ovarian cancers (e.g., tumors resistantto tamoxifen). The Grp94 inhibitors of the disclosure may be used incombination with an antiestrogen such as a selective estrogen receptormodulator (e.g., tamoxifen) or an aromatase inhibitor (e.g., exemestoneor anastrozole).

In some embodiments, the Grp94 inhibitors of the disclosure can be usedto treat EGFR dependent cancers that are resistant to therapy with EGFRinhibitors. In one such embodiment, the cancer is pancreatic cancer thatis resistant to therapy with EGFR inhibitors. The Grp94 inhibitor can beused in combination with an EGFR inhibitor. In particular embodiments, aGrp94 inhibitor is used in combination with the EGFR inhibitor erlotinibin the treatment of pancreatic cancer.

In other embodiments, the Grp94 inhibitors of the disclosure can be usedto treat Insulin growth factor 1 receptor (IGF1R) dependent tumors. Inparticular, the Grp94 inhibitors of the disclosure can be used intreating cancers with altered expression of the IGFIR where the receptoris necessary for pathogenesis and tumor progression. In a particularembodiment, the IGFIR dependent cancer is Ewing's sarcoma. In anotherparticular embodiment, the IGFIR dependent tumor is ovarian cancer.

The Grp94 inhibitors of the present disclosure can also be used to treatautoimmune diseases, inflammatory and neurodegenerative diseases,rheumatoid arthritis and diabetes. In some such embodiments, the Grp94inhibitors of the disclosure have an anti-angiogenic effect in type 1diabetes. In particular, the Grp94 inhibitors of the disclosure candisplay an anti-angiogenic effect on human endothelial cells.

The Grp94 inhibitors of the disclosure are capable of modulatinginflammatory responses through the inhibition of the Grp94 chaperoningof Toll-like receptors (TLRs), particularly TLR9. In particularembodiments, the Grp94 inhibitors of the disclosure can be used in thetreatment of inflammatory diseases such as lupus erythematosus,rheumatoid arthritis, ischemia reperfusion injury, atheroscleroticlesions, antibiotic associated colitis, and septic shock.

As described herein, the Grp94 inhibitors of the disclosure, whenprovided at a low enough dose, can be administered to cancer patientswithout a feed-back up-regulation of anti-apoptotic andresistance-mediating heat shock proteins, such as Hsp70. As such, theGrp94 inhibitors of the disclosure can be administered to patientswithout concomitant administration of an Hsp70 inhibitor. Hence, inaccordance with one aspect of the disclosure, methods of treating cancerby treating a human patient suffering from cancer without up-regulationof Hsp70 are provided. Such methods involve administration of a Grp94inhibitor of the disclosure in an amount sufficient to inhibit Grp94without inhibiting other Hsp90 paralogs (i.e., Hsp90α, Hsp90β and/orTrap-1). In one embodiment, a Grp94 inhibitor of the disclosure can beadministered to a cancer patient in an amount sufficient to inhibitGrp94 without inhibiting Hsp90α. In one embodiment, a Grp94 inhibitor ofthe disclosure can be administered to a cancer patient in an amountsufficient to inhibit Grp94 without inhibiting Hsp90β. In anotherembodiment, a Grp94 inhibitor of the disclosure can be administered to acancer patient in an amount sufficient to inhibit Grp94 withoutinhibiting TRAP-1. In another embodiment, a Grp94 inhibitor of thedisclosure can be administered to a cancer patient in an amountsufficient to inhibit Grp94 without up-regulation of Hsp70.

Furthermore, the Grp94 inhibitors of the disclosure are particularlyeffective in inducing apoptosis in cancer cells that overexpresstyrosine kinase receptors, particularly HER2 and EGFR. The ability ofthe Grp94 inhibitors to induce apoptosis stems in part from theinventors' discoveries that Grp94 has a role in maintaining high densityHER2 and EGFR species at the plasma membrane. The associated aberrantsignaling of these overexpressed proteins also requires Grp94. Thepresent invention encompasses the recognition that Grp94 inhibition ofHER2 and EGFR overexpressing tumors are highly sensitive to Grp94inhibition and readily undergo apoptosis upon administration of aselective Grp94 inhibitor. Accordingly, in one aspect, methods ofinducing apoptosis of HER2 and EGFR overexpressing tumors are providedby administration of a Grp94 inhibitor of the disclosure.

In another aspect the disclosure provides a versatile experimental assaythat can test rapidly and accurately the binding affinity of all majorHsp90 paralogs and has a testing range that spans low nanomolar tomillimolar binding affinities. The assay relies on using novelfluorescently labeled probes in fluorescence polarization (FP) assays.The fluorescently labeled probes, referred to herein as FP probes or FPtracers, are capable of binding to the four Hsp90 paralogs, Grp94,Hsp90α, Hsp90β and Trap-1, and therefore, can be used to determine theaffinity and selectivity of Hsp90 inhibitors to the four Hsp90 paralogs.Exemplary new FP probes are described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows structures of select Grp94 selective compounds and theirsubtype classification. FIG. 1B shows binding affinity of Grp94selective compound for the four Hsp90 paralogs. Data are presented asmean±s.d. (n=3). Values for PU-H71, a pan-Hsp90 inhibitor are presentedfor comparison. FIG. 1C shows Selectivity profile analysis for theselect ligands.

FIG. 2A-FIG. 2F show that PU-H54 unveils a novel drugable pocket inGrp94. FIG. 2A shows that Grp94 Apo adopts an “open” conformationsimilar to that observed in all Hsp90 N structures. FIG. 2B shows the“partially closed” conformation seen in the Grp94N:PU-H54 complex, whichis characterized by the incorporation of strand 1 into a longer helix 1and the downward rotation of helix 1 away from the core of the N-domain.Helices 4 and 5 reorient to straddle the repositioned helix 1. FIG. 2Cshows the “extended open” lid configuration seen in all nucleotide-boundstructures of Grp94N. Steric and electrostatic clashes contributed bythe phosphate moieties of the nucleotide cause the Helices 1, 4, and 5open up to fully expose ATP binding pocket. FIG. 2D shows the overlay ofHsp90- and Grp94-bound PU-H54 reveals an 80° torsional rotation aboutthe sulfanyl linker (highlighted in red) when inserted into theGrp94-specific channel. FIG. 2E shows the interactions of PU-H54 boundto Grp94 showing the increased hydrophobic stabilization of the 8-arylgroup when bound into Site 2. FIG. 2F shows a simple two-dimensionalschematic showing approximate locations of the amino acids of bindingsite 1 and binding site 2 of Grp94.

FIGS. 3A and 3B show PU-H54 bound to Site 1 of Hsp90a NTD. The purinescaffold maintains all previously observed purine-protein contacts, andthe 8-aryl group extends upwards into a hydrophobic channel betweenhelix 3 and the beta sheet core where it is sandwiched into Site 1,which is formed by the non-polar side chains of Leu107 on one side andPhe138 on the other. The pent-4-ynyl tail at the N3 position packsbeneath the purine ring, as has been observed previously for thissubstituent. The asymmetric 8-aryl group of PU-H54 adopts both thes-trans (25%) and s-cis (75%) configurations in the crystal structure,giving rise to a pseudosymmetric 8-aryl ring in the binding pocket. FIG.3C shows PU-H54 bound to Grp94. The structure of PU-H54 bound to Grp94shows that while the purine moiety of the ligand maintains contacts withconserved residues in the ATP pocket, the 8-aryl group adopts astrikingly different conformation compared to that of the Hsp90-boundPU-H54, specifically a “backwards” orientation. Concurrent with thisbackwards pose of the ligand, Phe199 of Grp94 swings away from thebinding pocket by 4 Å to expose a deep, almost completely hydrophobiccleft. The hydrophobic cleft is lined by binding site 2 amino acidsPhe195, Gly198, Val209, Ala202, Leu104, Leu249, and Phe203 as well aspart of binding site 1 amino acids Phe199, Ile247, Val211, Met154 andLeu163.

FIG. 4A-FIG. 4F show functionalities that confer Grp94- and Hsp90a/pselectivity: FIG. 4A and FIG. 4D show the general scheme portraying thetwo Grp94- and the two Hsp90α/β-selective ligand subtypes. FIG. 4B andFIG. 4C show interactions of the two Grp94-selective ligand subtypeswith the paralogs that confer selectivity and affinity for Grp94 andlessen binding to Hsp90α, Hsp90β and Trap-1. FIG. 4E and FIG. 4F showinteractions of the two Hsp90α/β-selective ligand subtypes with theparalogs that lessen binding to Grp94 and Trap-1 and confer selectivityand affinity for Hsp90α/β.

FIG. 5A-FIG. 5G show Grp94 and Hsp90α/β selective compounds exhibitselective paralog inhibition of IGF-II secretion by differentiated C2C12cells. FIG. 5A shows differentiated C2C12 cells were treated for 24 hrswith the indicated compounds. IGF-II secretion in the media from eachexperimental condition was measured and quantified against vehicle onlytreated cells (DMSO). Data are presented as mean±SEM (n=4). FIG. 5Bshows representative Western blot of cells as in FIG. 5A. Only pan-Hsp90inhibitors (geldanamycin (GM) and PU-H71) and the Hsp90 inhibitor(PU-29F) induce Hsp70 and degrade AKT, while the Grp94 inhibitor(PU-WS13) has no effect on these Hsp90-mediated functions. FIG. 5Cviability of C2C12 cells was visualized by light microscopy. Cells werefirst treated with or without the differentiation agent (2% horse serum)then added vehicle (DMSO) or the indicated concentrations of inhibitorsfor 24 h. The appearance of rounded, floating cells in the GM treatedconditions is indicative of cell killing. Representative images areshown. FIGS. 5D and 5F show trafficking of Toll-like receptor 9 (TLR9)to the cell surface. FIG. 5D (left) shows representative confocalmicroscopy image of HEK293 cells transfected with Empty vector orHA-TLR9 and stained as indicated. FIG. 5D (right) shows representativewestern blot confirming HA-TLR9 transfection of cells as indicated inthe left panel. FIG. 5E shows representative image and quantification ofquadruplicate experimental conditions of HEK293 cells transfected withHA-TLR9 (red) and treated for 24 h with the indicated concentrations ofPU-WS13 or PU-29F. Blue=DAPI. FIG. 5F shows representative image ofHEK293 cells transfected with HA-TLR9 (green) and treated for 24 h withthe indicated concentrations of PU-WS13 or PU-29F. Only the pan-Hsp90inhibitors (GM, PU-H71) both inhibit TLR9 trafficking and induce Hsp70.The Hsp90 inhibitor (PU-29F) fails to inhibit TLR9 trafficking while itinduces Hsp70. The Grp94 inhibitor (PU-WS13) inhibits TLR9 traffickingbut fails to induce Hsp70. FIG. 5G shows representative Western blot ofcells as in FIG. 5F.

FIG. 6A-FIG. 6F show that HER2 is sensitive to Hsp90 paralog inhibitionin a tumor-specific manner. FIG. 6A shows HER2 levels, quantified andnormalized, were plotted against the inhibitor concentration in SKBr3and MCF7 cells treated for 24 h with vehicle (DMSO) or the indicatedconcentrations of the Grp94-selective inhibitors PU-WS13 and PU-H39(top) or the Hsp90a/3-selective inhibitors PU-29F, PU-20F and PU-11(bottom). FIG. 6B shows the same as in FIG. 6A but for cells in whichGrp94 (top) or Hsp90a/o (bottom) was knocked-down by means of siRNA.FIG. 6C shows the same as in FIG. 6a but for HER2 and Raf-1 levels. Datafor Hsp90 paralog binding affinity is presented under each panel. FIGS.6A-C data are presented as mean±s.d. (n=3). FIG. 6D shows correlativeanalysis of Hsp90 paralog affinity versus HER2 degradation activity forselect compounds (n=7). Data were analyzed in GraphPad Prism software.FIG. 6E shows representative Western blots (WB) of HER2 complexes inMCF7 extracts isolated by precipitation with an anti-HER2 antibody or anonspecific IgG. FIG. 6F shows representative WB of MCF7 cells treatedfor the indicated times with PU-WS13 (15 μM) or PU-11 (40 μM). Proteinlevels in membrane and cytosolic fractions were plotted against the timeof treatment. Data are presented as mean±SEM (n=3).

FIG. 7A-1, FIG. 7A-2, and FIG. 7B-FIG. 7D show that HER2 is regulated bythe Hsp90 paralogs in a cellular compartment and a cell-specific manner.FIGS. 7A-1 and 7A-2 show that Grp94 inhibition leads to reducedsteady-state levels of HER2 in SKBr3 but not in MCF7 cells, whereasHsp90 inhibition downregulates HER2 in both SKBr3 and MCF7 cells. FIG.7A-1 shows representative western blot of SKBr3 and MCF7 cells treatedfor 24 h with the pan-Hsp90 inhibitor PU-H71 (1 μM), vehicle (DMSO),PU-WS13 (15 μM) or the indicated concentrations of the Grp94-selectiveinhibitors PU-WS13 and PU-H39. FIG. 7A-2 shows representative westernblot of SKBr3 and MCF7 cells treated for 24 h with the pan-Hsp90inhibitor PU-H71 (1 μM), vehicle (DMSO) or the indicated concentrationsof the Hsp90α/β selective inhibitor PU-29F, PU-20F and PU-11. FIG. 7Bshows that Grp94 knockdown leads to reduced steady-state levels of HER2in SKBr3 but not in MCF7 cells, whereas Hsp90 knockdown downregulatesHER2 in both SKBr3 and MCF7 cells. FIG. 7B (top) shows representativewestern blot of SKBr3 and MCF7 cells in which Grp94 was knocked-down bymeans of three distinct siRNAs generated against Grp94 or by a controlsiRNA (scramble). For comparison cell were also treated for 24 h withthe pan-Hsp90 inhibitor PU-H71 (1 μM), vehicle (DMSO) and the Grp94inhibitors PU-WS13 (15 μM) and PU-H39 (40 μM). FIG. 7B (bottom) showsrepresentative western blot of SKBr3 and MCF7 cells in which Hsp90 wasknocked-down by means of eight distinct siRNAs generated against theindicated Hsp90 paralogs or by a control siRNA (scramble). Forcomparison cell were also treated for 24 h with the pan-Hsp90 inhibitorPU-H71 (1 μM), vehicle (DMSO) and the Hsp90 inhibitors PU-29F (25 μM)and PU-20F (30 μM). FIG. 7C shows the same as in FIG. 7B but with Hsp90paralog levels being normalized to β-actin and changes in cytosolicHsp90 paralogs being graphed as “fold change”. Note feed-back inductionof Hsp90β upon Hsp90α knockdown. FIG. 7D shows fluorescence microscopyimage of SKBr3 cells treated for 4 h with DMSO, PU-WS13 (15 μM), PU-29F(20 μM) or PU-H71 (1 μM) and then stained with the indicated markersupon fixation and permeabilization. Inhibitor destabilized HER2co-localizes with endosomal structures adjoining the plasma membrane(for Grp94 inhibition) or with those found inside the cytosol (for Hsp90inhibition).

FIG. 8A-FIG. 8J show Grp94 and Hsp90 regulate distinct HER2 functions inHER2-overexpressing cancer cells. FIG. 8A shows representative flowcytometry of SKBr3 cells stained with a Grp94-specific antibody or anisotype control antibody shows cell surface localization of Grp94 thatis reduced by the protein trafficking inhibitor Brefeldin A. FIG. 8Bshows a fluorescence microscopy image of SKBr3 cells treated for 4 hwith DMSO or PU-WS13 (15 μM) and then stained with the indicated markersupon fixation and permeabilization. FIG. 8C shows representative blot ofsurface exposed proteins chemically labeled with biotin and purifiedusing streptavidin columns. Histone H4 was blotted to control formembrane impermeability. Total cell extracts; Total, Supernatant;non-surface proteins. Proteins eluted from the streptavidin column wereaffinity purified and analyzed by WB as indicated. FIG. 8D showsrepresentative WB of Grp94 and HER2 complexes isolated from plasmamembrane extracts (Fraction 5) as indicated. CP and IP, chemical andimmuno-precipitation, respectively. FIG. 8E shows representativeaffinity purification blot and the correlative analysis between Grp94and HER2 levels in complexes isolated from extracts in which Grp94levels were first reduced by IP with the indicated antibodies. FIG. 8Fand FIG. 8G show fluorescence microscopy images of SKBr3 cells treatedfor 4 h with vehicle or inhibitors and then stained with the indicatedmarkers upon fixation and permeabilization. FIG. 8H and FIG. 8I showrepresentative WB of SKBr3 cells treated for the indicated times with 20μM of PU-WS13 or PU-29F. Proteins in membrane and cytosolic fractionswere plotted against the time of treatment. Data are presented asmean±SEM (n=3). FIG. 8J shows schematic representation of changes inboth HER2 structure and function that occur at the plasma membrane ofSKBr3 cells upon Grp94 inhibition.

FIG. 9 shows schematic representation summarizing the tumor-specificregulation of HER2 by the Hsp90 paralogs. All epithelial cells containtwo copies of the HER2-encoding gene and express small amounts of theHER2 receptor on the cell surface. During oncogenic transformation, thenumber of gene copies per cell may increase, as in the SKBr3 cell line,leading to an increase in mRNA transcription and a 100- to 1,000-foldincrease in the number of HER2 receptors on the cell surface. Hsp90 issufficient for HER2 function in most cells with low to medium-HER2expression. Under conditions in which the stress imposed on the cell byproteome alterations (i.e. HER2 plasma overexpression) Grp94 also comesinto play, and, the chaperoning function of Grp94 is vital for properHER2 functioning in these conditions. Because HER2 is the major oncogenein these cells, its dependence on Grp94 renders cells addicted to properGrp94 functioning. Grp94 therefore becomes a target in such cancers.

FIG. 10A-FIG. 10F show Grp94 inhibition alone is sufficient to induceapoptosis in and reduce the viability of HER2 overexpressing breastcancer cells. FIG. 10A and FIG. 10B show viability of breast cancercells in which Grp94 was inhibited with PU-WS13 or knocked-down by meansof siRNA. Cell viability was assessed using an assay that quantifies ATPlevels. FIG. 10C shows cell killing (subG1 population) was determined inSKBr3 cells treated for the indicated times with PU-WS13 (15 μM). FIGS.10D and 10E show representative WB of cancer cells treated for 24 h withPU-WS13 or vehicle. FIG. 10F shows double staining with Annexin V and7AAD indicates induction of apoptosis following treatment of theindicated breast cancer cells for 48 h with PU-WS13 (10 μM).

FIG. 11A-FIG. 11D show Grp94 but not Hsp90 inhibition alone issufficient to induce death of HER2-overexpressing cells. FIGS. 11A and11B show representative western blots of HER2-overexpresssing cellstreated for 24 h with the pan-Hsp90 inhibitor PU-H71 (1 μM), vehicle(DMSO) or the indicated concentrations of the Grp94 selective inhibitorPU-WS13 or Hsp90α/β selective inhibitors PU-29F, PU-20F and PU-11.Cleaved PARP (cPARP) and cleaved caspase-3 (cCaspase-3) levels weremonitored to demonstrate induction of apoptosis or the lack of it.β-actin, loading control. Hsp70, specificity control. Hsp70 inductionfor Hsp90 inhibitors indicates inhibition of the cytosolic Hsp90 at thetested concentrations. Lack or minimal Hsp70 induction for Grp94inhibitors indicates no inhibition of the cytosolic Hsp90 at the testedconcentrations. FIG. 11C shows HER2++ breast cancer cells were treatedfor 72 h with the Hsp90α/β selective inhibitor PU-29F or the Grp94selective inhibitor PU-WS13 and cell viability was assessed using aviability assay that quantifies ATP levels. Y-axis values below zeroindicate killing of the initial cell population. FIG. 11D shows doublestaining with Annexin V and 7AAD indicates induction of apoptosisfollowing treatment of the SKBr3 HER2-overexpressing cells for 48 h withPU-WS13 (10 μM).

FIG. 12A shows the sensitivity of gastric cancers and esophageal cancercells to a selective Grp94 inhibitor. The OE19 and NCI-N87 cells, whichoverexpress high levels of HER2, were susceptible to Grp94 inhibition.The MNK74 cells, which do not overexpress HER2, were not susceptible toGrp94 inhibition. FIG. 12B shows double staining with Annexin V and 7AADindicates induction of apoptosis following treatment of the indicatedgastric and esophageal cancer cells for 48 h with PU-WS13 (10 μM).

FIG. 13A-FIG. 13C show that EGFR overexpressing triple negative breastcancer cells are sensitive to the selective Grp94 inhibitor PU-WS13. Thesensitivity of the EGFR overexpressing triple negative breast cancercells was tested for the presence of apoptotic cells double stainingwith Annexin V and 7AAD (FIGS. 13A, 13B) and by immunoblotting for thepresence of cleaved PARP (FIG. 13C).

FIG. 14A and FIG. 14B show that EGFR overexpressing cancer cells aresensitive to the Grp94 selective inhibitor PU-WS13. FIG. 14A shows thatthe selective Grp94 inhibitor PU-WS13 effectively inhibited the growthof the EGFR overexpressing PANC-1 cells but had no effect on the Capan-2cells and a modest effect on the growth of the CFPAC cells (FIG. 14A).FIG. 14B shows there was a substantial increase in cells exhibitingmarkers of early- and late-stage apoptosis observed for the PANC-1 cellsbut not for the Capan-2 cells, as indicated by double staining withAnnexin V and 7AAD.

FIG. 15 shows that treatment of EGFR-overexpressing PANC-1 cells withthe Grp94 selective inhibitor PU-WS13 was more potent at killing cellsthrough apoptosis than were the pan-HSP90 inhibitor PU-H71 and theHSP90α inhibitor PU-29F. Double staining with Annexin V and 7AADindicates induction of apoptosis.

FIG. 16A and FIG. 16B show that the Grp94 selective inhibitor PU-WS13induces apoptosis in IGF1R overexpressing Ewing sarcoma cell lines (FIG.16A). Double staining with Annexin V and 7AAD indicates induction ofapoptosis (FIG. 16B).

FIG. 17 shows that the Grp94 selective inhibitor PU-WS13 inducesapoptosis in IGF1R and TGFbeta expressing ovarian cancer cell linesderived from a poorly differentiated serous adenocarcinoma.

FIG. 18A and FIG. 18B show that the angiogenic effect of both nativeGrp94 and the IgG-containing fraction purified from plasma of diabeticsubjects, referred to as peak 2 (p2mQ) is inhibited by the Grp94inhibitor PU-H54 (FIG. 18A). Grp94 promotes the angiogenictransformation of Human Umbilical Vein Endothelial Cells (HUVECs) by acytokine-like mechanism. Overall, the morphologic changes observed inthe presence of PU-H54 demonstrate that Grp94 inhibition displays ananti-angiogenic effect on HUVECs while it does not affect substantiallycell proliferation (FIG. 18B).

FIG. 19A and FIG. 19B show that the Grp94 selective inhibitors PU-WS13(FIG. 19A) and PU-H54 (FIG. 19B) inhibit TLR9 ligand, CpG DNA, inducedTNF-α production in mouse macrophages.

FIG. 20 shows the chemical structure of Compound 40.

FIG. 21A shows the strategy for designing FP probes based on the Hsp90inhibitor PU-H71. FIG. 21B shows probe 43a docked into the HSP90α ATPbinding pocket (PDB ID: 2FWZ) as generated by Glide (version 5.0).Modeling shows potential steric clach between the probe and Leu107 forlinkers containing less than 3 carbons.

FIG. 22A-FIG. 22C show the dose-response curve for the binding ofindicated probes to the Hsp90 paralogs from a cancer cell extract (FIG.22A) or to individual Hsp90 paralogs (FIG. 22B-FIG. 22C). Differentamounts of protein (were incubated with the ligand at 4° C. and theresponse measured at equilibrium (24 h). The assay window data wereobtained by subtracting free probe values from values recorded in thepresence of specified protein concentrations. Data were analyzed andplotted in Prism 4.0. Average values from duplicate experiments arepresented.

FIG. 23 shows the structures of known Hsp90 inhibitors which wereanalyzed using fluorescence polarization methods of the disclosure.

FIG. 24A shows Western blot analysis of the activity of PU-WS13 in apanel of cancer cells; ( ) FIG. 24B MDA-MB-468 cells were treated for 24h with the indicated concentration of PU-WS13 or with vehicle (-); (FIG.24C) HMEC cells were treated for 24 h with the indicated concentrationof PU-WS13, PU29F or with vehicle (-). The expression of HER2 and EGFR,as well as the expression and activity of proteins involved indownstream signaling through these receptors (STAT, AKT ERK) wereanalyzed by Western blot.

FIG. 25A-FIG. 25D show a PK/PD analysis for PU-WS13 tumor retention andselective targeting of Grp94 cancer functions. Mice bearing tumors wereinjected ip 75 mg/kg of PU-WS13. Mice were sacrificed at the indicatedtimes post-PU-WS13 injection and tissues, tumors, and plasma wereharvested. PU-WS13 levels were analyzed by LCMSMS in the indicatedtumors (FIG. 25A) or tissues (FIG. 25C). Proteins in the indicatedtumors (FIG. 25B) or tissues (FIG. 25D) were analyzed by Western blot.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides, among other things, Grp94 selectiveinhibitors. These Grp94 selective inhibitors are capable of inhibitingGrp94 without inhibiting the other Hsp90 paralogs, including Hsp90π,HSP90β and Trap-1. Accordingly, the Grp94 inhibitors of the disclosurecan antagonize the chaperone function of Grp94 without inhibiting thechaperone function of the other Hsp90 paralogs, including Hsp90α, HSP90βand Trap-1. The compounds of the disclosure can be used in therapeuticmethods by administering a therapeutically effective amount of acompound of the disclosure to an individual, including a human, in needof treatment for cancer, a neurodegenerative disease, an autoimmunedisease, an inflammatory disease or other condition for which Grp94inhibition is relevant. In particular embodiments, the Grp94 inhibitorsof the disclosure can be administered at a dosage that inhibits Grp94without inhibiting the biological activity (e.g., chaperone function) ofHsp90π, HSP90β and/or Trap-1.

As used in this application, the term “treatment” refers to delaying theonset of symptoms, reducing the severity or delaying the symptomaticprogression of cancer, neurodegenerative disease or other condition inthe individual. A cure of the disease is not required to fall within thescope of treatment. Further, it will be appreciated that the specificresults of these treatment goals will vary from individual toindividual, and that some individuals may obtain greater or lesserbenefits than the statistical average for a representative population.Thus, treatment refers to administration of composition to an individualin need, with the expectation that they will obtain a therapeuticbenefit.

The term “administering” refers to the act of introducing into theindividual the therapeutic compound. In general, any route ofadministration can be used. Thus, administration by oral, intrathecal,intravenous, intramuscular or parenteral injection is appropriatedepending on the nature of the condition to be treated. Administrationmay also be done to the brain by inhalation because there is acompartment at the upper side of the nose that connects with the brainwithout having the blood brain barrier capillaries. Compounds that donot cross the blood brain barrier are preferred for this mode ofadministration, although this characteristic is not strictly required.

The term “therapeutically effective amount” encompasses both the amountof the compound administered and the schedule of administration that ona statistical basis obtains the result of preventing, reducing theseverity or delaying the progression of the disease in the individual.As will be appreciated, preferred amounts will vary from compound tocompound in order to balance toxicity/tolerance with therapeuticefficacy and the mode of administration.

5.1 Identification of Grp94 Selective Binding Site

To identify paralog specific Hsp90 inhibitors, an in-house generatedlibrary of over 130 purine-scaffold (PU)-compounds in a fluorescencepolarization (FP) based assay to test for binding to recombinant Hsp90aand Grp94. FP methods described herein make use of fluorescently labeledprobes (tracers) that bind to the different Hsp90 paralogs. Thus, oneaspect of the present invention is the provision of fluorescentlylabelled Grp94 inhibitors, wherein any compound described herein isderivatized with a fluorescent label. Methods of making such labelledcompounds are described herein and in International Patent PublicationNo. WO/2013/009657, the entire contents of which is hereby incorporatedby reference. The present invention also encompasses radiolabelledanalogs of provided compounds. Methods of making such radiolabelledcompounds are known in the art, for example in International PatentPublication No. WO/2013/009655, the entire contents of which is herebyincorporated by reference.

Potential inhibitors of the respective paralogs are determined bymeasuring the ability of the inhibitor to disrupt binding of thefluorescently labeled probe to the specific Hsp90 paralog. The presentinvention provides a series of new fluorescently labeled probes aredescribed that can bind to all four Hsp90 paralogs. Accordingly,competition assays can be conducted using a single fluorescently labeledprobe for each of the different Hsp90 paralogs being analyzed.Alternatively, more than one labeled probe can be used in the bindingassays. For instance, the probe Cy3B-GM was used in determining thebinding of small molecule inhibitors to Hsp90π, Hsp90β and Grp94 whilethe fluorescently labeled probe PU-FITC3 was used in determining thebinding of small molecule inhibitors to Trap-1. The structures ofCy3B-GM and PU-FITC3 are shown below:

Select derivatives were also analyzed for binding to Hsp90β and Trap-1.The purine-scaffold library was designed with bias for binding to theHsp90 Bergerat-type pockets. As expected from the high analogy in theHsp90 ATP-binding pockets, a majority of the tested compounds exhibitedsimilar affinities for the two paralogs and comprised chemical spaces oflittle or no selectivity. Nonetheless, a chemical space with selectivityfor Grp94 was identified. The structures of these compounds as well astheir binding affinity to the different paralogs of HSP90 are shown inFIG. 1. Importantly, select compounds of the Grp94 selective chemicalspace exhibited greater than 100-fold preference for Grp94 over Hsp90α/βand a 10- to 100-fold preference over Trap-1.

Despite the strong Grp94 selectivity uncovered in the screeningexperiments, modeling of these compounds into the ATP binding pockets ofexisting structures of Grp94 and Hsp90 did not reveal significantdifferences that could account for the observed binding selectivity.Therefore, the structure of the Grp94-specific ligand PU-H54 in complexwith the NTD fragment of both Grp94 and human Hsp90a (Grp94N and Hsp90NTD, respectively) was determined (FIGS. 2 and 3). In agreement withprevious crystal structures, the tertiary structure of Hsp90 in complexwith PU-H54 was essentially identical to that of all otherhHsp90N-ligand complexes (Immormino, R. M., Kang, Y., Chiosis, G. &Gewirth, D. T. Structural and quantum chemical studies of8-aryl-sulfanyl adenine class Hsp90 inhibitors. J. Med. Chem. 49,4953-4960 (2006)) (FIG. 2a, b ). While inserted into this pocket, PU-H54lacks X2-Ar substituents that would confer it strong binding, providingan explanation for the low affinity of this ligand for Hsp90 (FIG. 1b ).

In the structure of the Grp94:PU-H54 complex, on the other hand, thehelix 1,4,5 “lid” region in Grp94 adopted a novel “partially closed”conformation, whereby strand 1 and helix 1 were pulled away from thecore of the N domain, and helices 4 and 5 shifted upwards to straddlethe top of helix 1 (FIG. 2a-c ). These rearrangements also repositionedhelix 3 of Grp94, resulting in a slightly larger ATP binding pocket. Thestructure of PU-H54 bound to Grp94 showed that while the purine moietyof the ligand maintained contacts with conserved residues in the ATPpocket (FIG. 2c ), the 8-aryl group adopted a strikingly differentconformation compared to that of the Hsp90-bound PU-H54 (FIG. 2d ).Overlaying the Hsp90- and Grp94-bound PU-H54 ligands revealed an ˜80°torsional rotation of the 8-aryl group about the sulfanyl linker, wherethe Hsp90-bound ligand adopted the “forward” rotation, and theGrp94-bound ligand adopted a novel “backwards” rotation (FIG. 2d ).Concurrent with this backwards pose of the ligand, Phe199 of Grp94swings away from the binding pocket by 4 Å to expose a deep, almostcompletely hydrophobic cleft (FIG. 2e —Not all amino acid residues ofthe binding site are depicted).

For convenience of description, we have divided the hydrophobic cleftinto two distinct binding sites referred to as “binding site 1” and“binding site 2” of the NTD of Grp94. The full length sequence of humanGrp94 is shown as SEQ ID NO: 1 in Table 1, below. See also U.S. Pat.Nos. 7,991,601 and 7,589,174. The sequence of the N-terminal Domain ofhuman Hsp90a (amino acids 1-236) is shown as SEQ ID NO:2 in Table 1. Thesequence of the N-terminal Domain of human Hsp90β (amino acids 1-233) isshown as SEQ ID NO:3 in Table 1. The full length sequence of humanTRAP-1 is shown as SEQ ID NO:4 in Table 1. A simple two-dimensionalschematic showing approximate locations of the amino acids of bindingsite 1 and binding site 2 is shown in FIG. 2f . Binding site 1 is linedby at least the amino acids Ile247, Val211, Phe199, Met154 and Leu163 ofSEQ ID NO: 1. Binding site 1 can also include the amino acids Leu159,Trp223, and Tyr200 of SEQ ID NO: 1 (not shown). Notably, interaction ofligands (e.g., ATP or small molecule inhibitors) with the amino acidscomprising binding site 1 are conserved in Hsp90π, Hsp90β, and Trap-1.Binding site 2 is lined by the amino acids Phe195, Gly198, Val209,Ala202, Phe203, Leu104, and Leu249 of SEQ ID NO: 1. A similar cavitycomposed of the equivalent, conserved, residues of binding site 2 isalso present in the other Grp94 paralogs but access to binding site 2 isblocked by Phe138 in Hsp90a and Hsp90β, and Phe205 in Trap-1.Accordingly, binding site 2 of SEQ ID NO: 1 is a Grp94 specific bindingsite. The hydrophobic X2-Ar of Grp94-bound PU-H54 is inserted into thisnewly-revealed non-polar binding site 2 and makes stabilizing contactswith at least 5 of its residues. In FIG. 2f , the residues Leu159,Tyr200, and Trp223 (marked in triangles) do not interact with the Grp94selective inhibitors of the disclosure. However, these residues arecapable of interacting with pan-Hsp90 inhibitors (e.g., PU-H71).Notably, residues Asp149, Asn107, Thr245, Ala111, Gly153, Ala108, andLys114 (FIG. 2f ) are conserved in all Hsp90 paralogs. Accordingly, thepurine portion of pan-Hsp90 inhibitors and selective Grp94 inhibitors ofthe disclosure interact with these residues.

TABLE 1 Sequence of Human Hsp90 paralogs SEQ ID NO. Description Sequence1 Grp94 MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSR SequenceEGSRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQA (Human)EVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNAKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPAKVEEEPEEEPEETAEDTTEDTEQD EDEEMDVGTDEEEETAKESTAEKDEL 2Hsp90α MGSSHHHHHHSSGLVPRGSHMPEETQTQDQPMEEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNSSDALDKIRYESLTDPSKLDSGKELHINLIPNKQDRTLTIVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVTVITKHNDDEQYAWESSAGGSFTVRTDTGEPMGRGTVILHLKEDQTEYLEERRIKEIVKKHSQFIGYPITLFVEKERDKE VSDDEAE 3 Hsp90βDPTLMPEEVHHGEEEVETFAFQAEIAQLMSLIINTFYSNKEIFLRELISNASDALDKIRYESLTDPSKLDSGKELKIDIIPNPQERTLTLVDTGIGMTKADLINNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVAEKVVVITKHNDDEQYAWESSAGGSFTVRADHGEPIGRGTKVILHLKEDQTEYLEERRVKEVVKKHSQFIGYPITLYLEKEREKGEFNSKLGCFGG 4 TRAP-1MARELRALLLWGRRLRPLLRAPALAAVPGGKPILCPRRTTAQLGPRRNPAWSLQAGRLFSTQTAEDKEEPLHSIISSTESVQGSTSKHEFQAETKKLLDIVARSLYSEKEVFIRELISNASDALEKLRHKLVSDGQALPEMEIHLQTNAEKGTITIQDTGIGMTQEELVSNLGTIARSGSKAFLDALQNQAEASSKIIGQFGVGFYSAFMVADRVEVYSRSAAPGSLGYQWLSDGSGVFEIAEASGVRTGTKIIIHLKSDCKEFSSEARVRDVVTKYSNFVSFPLYLNGRRMNTLQAIWMMDPKDVREWQHEEFYRYVAQAHDKPRYTLHYKTDAPLNIRSIFYVPDMKPSMFDVSRELGSSVALYSRKVLIQTKATDILPKWLRFIRGVVDSEDIPLNLSRELLQESALIRKLRDVLQQRLIKFFIDQSKKDAEKYAKFFEDYGLFMREGIVTATEQEVKEDIAKLLRYESSALPSGQLTSLSEYASRMRAGTRNIYYLCAPNRHLAEHSPYYEAMKKKDTEVLFCFEQFDELTLLHLREFDKKKLISVETDIVVDHYKEEKFEDRSPAAECLSEKETEELMAWMRNVLGSRVTNVKVTLRLDTHPAMVTVLEMGAARHFLRMQQLAKTQEERAQLLQPTLEINPRHALIKKLNQLRASEPGLAQLLVDQIYENAMIAA GLVDDPRAMVGRLNELLVKALERH

5.2 Ligand Characteristics that Confer Grp94 Selectivity

We next analyzed the functionalities that, when attached onto thepurine-scaffold, confer Grp94-specific binding. On close inspection, theGrp94 selective inhibitors could be classified into two structuralsubtypes: Ar-X2- and X3-dependent (FIG. 1a and FIG. 4a ). In theAr-X2-dependent subtype, we identified compounds of high bindingaffinity for Grp94 (FIG. 1b ) and also remarkable selectivity (greaterthan 100-fold) for Grp94 over Hsp90α/β and Trap-1. Energy minimizationsindicated that a subset of these compounds preferred the backwards bentconformation even in the unbound state. In addition, the preferentialpresence of hydrophobic substituents in the Grp94 selective binding sitewas observed. These allow for favorable proximity to the side chains ofbinding site 2 and binding site 1 (see FIG. 2f ) of Grp94. These matchedhydrophobic interactions provide a rationale for the preferentialpresence of these groups on the Grp94 selective ligands. Notably, theHsp90α/β and Trap-1 paralogs were unable to accommodate thesederivatives due to unfavorable interactions with several pocket residues(FIG. 4c , top).

In the X3-dependent subtype, the presence of a methyl group at theC1′-position of the N9 alkyl chain also yielded compounds with more than10-fold selectivity for Grp94 over Hsp90α/β and Trap-1. Molecularmodeling indicated that the C1′ methyl group favored the placement ofthe 8-aryl ring into the backwards bent conformation, resulting inbinding into Site 2 of Grp94 (FIG. 4b , bottom). In contrast to theAr-X2-dependent subtype described above, the affinity of these compoundsfor Grp94 was modest (60-90 μM), reflecting the less hydrophobiccharacter of the X2-substituents (i.e. trimethoxy). Hsp90α/β and Trap-1could not accommodate these inhibitors, potentially due to unfavorableinteractions between the C1′ methyl and substituents on the 8-aryl ringand between the ligand and several pocket residues (FIG. 4c , bottom).

Accordingly, in the purine-scaffold series, a two-log selectivity forGrp94 over other Hsp90 paralogs and a favorable affinity is limited tothose compounds that favor or may accommodate the “backwards”conformation and feature an aryl ring with hydrophobic substituents onthe 2′, 3′, 4′ and/or 5′ positions in the configuration described above.Both characteristics portend favorable interactions of the ligand withSite 2 of Grp94.

Based on the foregoing, novel Grp94 inhibitors with scaffolds based onpurine were identified based on their ability to accommodate the“backwards” conformation and to make favorable hydrophobic contacts withthe amino acids lining binding site 1 and binding site 2 of Grp94.Accordingly, in one aspect, the disclosure provides new compounds thatexhibit affinity for Grp94 and thus, are capable of inhibiting thebiological activity of Grp94. In some embodiments, the Grp94 inhibitorsinteract with six or more of the amino acids comprising binding site 1and binding site 2 of the Grp94 NTD. In particular embodiments, theGrp94 inhibitors of the disclosure can interact with six, seven, eight,nine, ten, eleven or twelve of the amino acids comprising binding site 1and binding site 2 of the Grp94 NTD. In other embodiments, the Grp94inhibitors of the disclosure interact with six or more amino acidsselected from Phe195, Gly198, Val209, Ala202, Ile247, Leu249, Phe203,Leu104, Val211, Phe199, Met154 and Leu163 of SEQ ID NO: 1. For instance,the Grp94 inhibitors of the disclosure can interact with six, seven,eight, nine, ten, eleven or twelve of the amino acids selected fromPhe195, Gly198, Val209, Ala202, Ile247, Leu249, Phe203, Leu104, Val211,Phe199, Met154 and Leu163 of SEQ ID NO. 1.

In particular embodiments, the Grp94 inhibitors of the disclosure arecapable of interacting with three or more of the amino acids in bindingsite 2 (i.e., the Grp94 selective binding site) of the Grp94 NTD. Forinstance, the Grp94 inhibitors can interact with three, four, five, sixor seven of the amino acids of binding site 2 of the Grp94 NTD. In somesuch embodiments, the Grp94 inhibitors of the disclosure are capable ofinteracting with three or more amino acids selected from Phe195, Gly198,Val209, Ala202, Leu104, Leu249 and Phe203 of SEQ ID NO: 1. For instance,the Grp94 inhibitors of the disclosure can interact with three, four,five, six or seven amino acids selected from Phe195, Gly198, Val209,Ala202, Leu104, Leu249 and Phe203 of SEQ ID NO:1.

In some embodiments, the Grp94 selective inhibitors of the disclosureare capable of interacting with the amino acids Ala202, Leu104 andLeu249 of SEQ ID NO: 1. In other embodiments, the Grp94 selectiveinhibitors of the disclosure are capable of interacting with the aminoacids Gly198, Val209, Ala202, Leu249 and Phe203 of SEQ ID NO: 1. Inother embodiments, the Grp94 selective inhibitors of the disclosure arecapable of interacting with the amino acids Phe195, Val209, Ala202 ofSEQ ID NO: 1. In other embodiments, the Grp94 selective inhibitors ofthe disclosure are capable of interacting with the amino acids Leu104,Val209, Ala202 of SEQ ID NO: 1. In still other embodiments, the Grp94selective inhibitors of the disclosure are capable of interacting withthe amino acids Phe195, Leu249 and Leu104 of SEQ ID NO: 1. In stillother embodiments, the Grp94 selective inhibitors of the disclosure arecapable of interacting with the amino acids Phe195, Gly198 and Val209 ofSEQ ID NO:1. In still other embodiments, the Grp94 selective inhibitorsof the disclosure are capable of interacting with the amino acidsLeu104, Leu249 and Phe203 of SEQ ID NO: 1.

5.3 Grp94 Inhibitors with Scaffolds Based on Purine

In one aspect, the disclosure provides selective Grp94 inhibitors withscaffolds related to purine (e.g., fused amino pyridine compounds). Insome embodiments, the Grp94 inhibitors are adenine scaffold inhibitors.In some embodiments, the Grp94 inhibitors are adenine scaffoldinhibitors.

In particular embodiments, the purine-scaffold (e.g., adenine-scaffold)inhibitors can be substituted at 8-position with a linker group bondedto an aryl or heteroaryl group. For instance, the substituent bonded tothe 8-position of the purine ring can be an arylsulfanyl group, anarylsulfoxyl group, an arylsulfonyl group, a benzyl group, an anilinegroup, an arylcarbonyl group, or a phenoxy group. In some suchembodiments, the aryl or heteroaryl group at the 8-position of thepurine ring interact with amino acids comprising binding site 1 andbinding site 2 of SEQ ID NO: 1. For instance, the aryl or heteroarylgroup at the 8-position of the purine ring can interact with six, seven,eight, nine, ten, eleven or twelve of the amino acids selected fromPhe195, Gly198, Val209, Ala202, Ile247, Leu249, Phe203, Leu104, Val211,Phe199, Met154 and Leu163 of SEQ ID NO. 1. In other embodiments, thearyl or heteroaryl group at the 8-position of the purine ring caninteract with three, four, five, six or seven amino acids selected fromPhe195, Gly198, Val209, Ala202, Ile247, Leu249 and Phe203 of SEQ IDNO: 1. The purine portion of the purine-scaffold Grp94 inhibitors of thedisclosure generally interacts with amino acids that are conserved inall Hsp90 paralogs. For instance, the purine portion can form favorableinteractions with Asp149, Thr245, Ala111, Gly153, Lys114, Ala108 andAsn107 of SEQ ID NO:1.

Owing to the hydrophobic nature of the intermolecular interactionsresponsible for binding of ligands to the Grp94 receptor, developingwater soluble inhibitors that have a desired level of cell permeabilityposed a challenge. Surprisingly, we have discovered that byspecification modifications of the functionality at the N-9 or N-3position of the purine scaffold, water soluble inhibitors that retaintheir high selectivity for Grp94 over the other Hsp90 paralogs can bedeveloped. Hence, in particular embodiments, the purine-scaffold Grp94inhibitors of the disclosure are water soluble. For instance, the watersolubility of the purine-scaffold inhibitors of the disclosure can begreater than 0.5 mg/mL at neutral pH and ambient temperatures. Forinstance, the water solubility of the purine-scaffold inhibitors of thedisclosure can be greater than 0.5 mg/mL, greater than 1 mg/mL, greaterthan 2 mg/mL, greater than 1 mg/mL, 2 mg/mL, 3 mg/mL, greater than 4mg/mL, greater than 5 mg/mL, greater than 6 mg/mL, greater than 10mg/mL, greater than 15 mg/mL, greater than 20 mg/mL, greater than 25mg/mL, greater than 30 mg/mL, greater than 40 mg/mL or greater than 50mg/mL in distilled water at ambient temperatures.

In embodiments where Grp94 inhibitors of the disclosure are onlyslightly soluble or insoluble, the inhibitors can be formulated in avehicle that increases their solubility. For instance, the Grp94inhibitors of the disclosure can be delivered in a vesicle, inparticular a liposome.

In all of the compounds of the present disclosure, the compound may beas depicted, or as a pharmaceutically acceptable salt thereof. In oneembodiment, the pharmaceutically acceptable salt is ahydrochloride-salt, a phosphate salt, a sulfate-salt, a citrate salt, anoxalate salt, a benzene sulfonic acid-salt, a para-toluenesulfonicacid-salt, a mesylate salt, a tartrate salt, a lactobionate salt, asuccinate salt or a fumaric acid-salt. In another embodiment, thepharmaceutically acceptable salt is a hydrochloride-salt or asulfate-salt. In another embodiment, the pharmaceutically acceptablesalt is a hydrochloride-salt. In another embodiment, thepharmaceutically acceptable salt is a sulfate-salt. In anotherembodiment, the pharmaceutically acceptable salt is a phosphate-salt.

In naming options for X¹, X², X³, X⁴, X⁵, X⁶, Y, Q, Z¹, Z², Z³, Z⁴, Z⁵,Z⁶, Z⁷, Z⁸ R¹, R², R³, R⁴, R⁷, and R⁸ the name refers to the type ofgroup that is directly attached to the central structure, which groupmay include additional functionality. Thus, “alkyl” group refers to alinear, cyclic or branched saturated hydrocarbon, for example ahydrocarbon having from 1 to 10 carbon atoms, in which the atom directlyattached to the central structure is a carbon atom. Such an alkyl groupmay include substituents other than hydrogen, for example anoxygen-containing group including without limitation hydroxyl andalkoxy; a halogen group; a nitrogen-containing group including withoutlimitation amino, amido and alkylamino; an aryl group; asulfur-containing group including without limitation thioalkyl; and/or anon-aromatic cyclic group including heterocycles and carbocycles. Carbonatoms in these substituents may increase the total number of carbonatoms in the alkyl group to above 10 without departing from theinvention. All references to alkyl groups in the specification andclaims hereof encompass both substituted and unsubstituted alkyl groupsunless the context is clearly to the contrary.

“Aliphatic” or “aliphatic group”, as used herein, means a straight-chain(i.e., unbranched) or branched, substituted or unsubstituted hydrocarbonchain that is completely saturated or that contains one or more units ofunsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon thatis completely saturated or that contains one or more units ofunsaturation, but which is not aromatic (also referred to herein as“carbocycle,” “carbocyclic”, “cycloaliphatic” or “cycloalkyl”), that hasa single point of attachment to the rest of the molecule. Unlessotherwise specified, aliphatic groups contain 1-6 aliphatic carbonatoms. In some embodiments, aliphatic groups contain 1-5 aliphaticcarbon atoms. In other embodiments, aliphatic groups contain 1-4aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodiments,aliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “carbocyclic” (or “cycloaliphatic” or “carbocycle” or“cycloalkyl”) refers to a monocyclic C₃-C₈ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

“Alkenyl” group refers to a linear, cyclic or branched hydrocarbon, forexample a hydrocarbon having from 1 to 10 carbon atoms, and at least onedouble bond, in which the atom directly attached to the centralstructure is a carbon atom. The alkenyl group may include any of thesubstituents mentioned above for an alkyl group. All references toalkenyl groups in the specification and claims hereof encompass bothsubstituted and unsubstituted alkenyl groups unless the context isclearly to the contrary.

“Alkynyl” group refers to a linear, cyclic or branched hydrocarbon, forexample a hydrocarbon having from 1 to 10 carbon atoms, and at least onetriple bond, in which the atom directly attached to the centralstructure is a carbon atom. The alkynyl group may include any of thesubstituents mentioned above for an alkyl group. All references toalkynyl groups in the specification and claims hereof encompass bothsubstituted and unsubstituted alkynyl groups unless the context isclearly to the contrary.

“Aryl” group refers to any group derived from a simple aromatic ring.Aryl group includes heteroaryl. An aryloxy substituent is an aryl groupconnected to the central structure through an oxygen atom. The arylgroup may include any of the substituents mentioned above for an alkylgroup, and in addition an aryl group may include an alkyl, alkenyl oralkynyl group. All references to aryl groups in the specification andclaims hereof encompass both substituted and unsubstituted aryl groupsunless the context is clearly to the contrary.

“Arylalkyl” refers to alkyl groups in which a hydrogen atom has beenreplaced with an aryl group. Such groups include, without limitation,benzyl, cinnamyl, and dihyrocinnamyl.

“Amino” group refers to any group which consists of a nitrogen attachedby single bonds to carbon or hydrogen atoms. In certain instances, thenitrogen of the amino group is directly bound to the central structure.In other instances, an amino group may be a substituent on or within agroup, with the nitrogen of the amino group being attached to thecentral structure through one or more intervening atoms. Examples ofamino groups include NH₂, alkylamino, alkenylamino groups andN-containing non-aromatic heterocyclic moiety (i.e., cyclic amines).Amino groups may be substituted or unsubstituted. All references toamino groups in the specification and claims hereof encompasssubstituted and unsubstituted amino groups unless the context is clearlyto the contrary.

“Halogen” group refers to fluorine, chlorine, bromine or iodine.

“Heterocyclic” group refers to a moiety containing at least one atom ofcarbon, and at least one atom of an element other than carbon, such assulfur, oxygen or nitrogen within a ring structure. These heterocyclicgroups may be either aromatic rings or saturated and unsaturatednon-aromatic rings. Heterocyclic groups may be substituted orunsubstituted. All references to heterocyclic groups in thespecification and claims encompass substituted and unsubstitutedheterocyclic groups unless the context is clearly to the contrary.

“—(C₃-C₈)cycloalkyl” refers to a saturated monocyclic hydrocarbon having3, 4, 5, 6, 7, or 8 carbon atoms. Representative (C₃-C₈)cycloalkylsinclude -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclohexyl,-cycloheptyl, and -cyclooctyl.

“—(C₃-C₈)heterocycloalkyl” refers to a saturated monocyclic hydrocarbonhaving 3, 4, 5, 6, 7, carbon atoms and one heteroatom independentlyselected from nitrogen, oxygen, and sulfur.

“-(5- or 6-membered)heteroaryl” refers to a monocyclic aromaticheterocycle ring of 5 or 6 members, i.e., a monocyclic aromatic ringcomprising at least one heteroatom independently selected from nitrogen,oxygen, and sulfur. In one embodiment, the -(5- or 6-membered)heteroarylring contains at least one carbon atom. Representative -(5- or6-membered)heteroaryls include pyridyl, furyl, pyrrolyl, oxazolyl,imidazolyl, thiazolyl, isoxazolyl, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,5-oxadiazolyl, 1,2,3-triazolyl, pyrazolyl, isothiazolyl,pyridazinyl, pyrimidyl, pyrazinyl, 1,2,3-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,5-triazinyl, and thiophenyl.

As used herein, the term “detectable moiety” is used interchangeablywith the term “label” and “reporter” and relates to any moiety capableof being detected, e.g., primary labels and secondary labels. A presenceof a detectable moiety can be measured using methods for quantifying (inabsolute, approximate or relative terms) the detectable moiety in asystem under study. In some embodiments, such methods are well known toone of ordinary skill in the art and include any methods that quantify areporter moiety (e.g., a label, a dye, a photocrosslinker, a cytotoxiccompound, a drug, an affinity label, a photoaffinity label, a reactivecompound, an antibody or antibody fragment, a biomaterial, ananoparticle, a spin label, a fluorophore, a metal-containing moiety, aradioactive moiety, quantum dot(s), a novel functional group, a groupthat covalently or noncovalently interacts with other molecules, aphotocaged moiety, an actinic radiation excitable moiety, a ligand, aphotoisomerizable moiety, biotin, a biotin analog (e.g., biotinsulfoxide), a moiety incorporating a heavy atom, a chemically cleavablegroup, a photocleavable group, a redox-active agent, an isotopicallylabeled moiety, a biophysical probe, a phosphorescent group, achemiluminescent group, an electron dense group, a magnetic group, anintercalating group, a chromophore, an energy transfer agent, abiologically active agent, a detectable label, and any combination ofthe above).

Primary labels, such as radioisotopes (e.g., tritium, ³²P, ³³P, ³⁵S,¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I), mass-tags including, but not limitedto, stable isotopes (e.g., ³C, ²H, ¹⁷O, ¹⁸O, ¹⁵N, ¹⁹F, and ¹²⁷I),positron emitting isotopes (e.g., ¹¹C, ¹⁸F, ¹³N, ¹²⁴I, and ¹⁵O), andfluorescent labels are signal generating reporter groups which can bedetected without further modifications. Detectable moities may beanalyzed by methods including, but not limited to fluorescence, positronemission tomography, SPECT medical imaging, chemiluminescence,electron-spin resonance, ultraviolet/visible absorbance spectroscopy,mass spectrometry, nuclear magnetic resonance, magnetic resonance, flowcytometry, autoradiography, scintillation counting, phosphoimaging, andelectrochemical methods.

The term “secondary label” as used herein refers to moieties such asbiotin and various protein antigens that require the presence of asecond intermediate for production of a detectable signal. For biotin,the secondary intermediate may include streptavidin-enzyme conjugates.For antigen labels, secondary intermediates may include antibody-enzymeconjugates. Some fluorescent groups act as secondary labels because theytransfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal.

The terms “fluorescent label”, “fluorescent dye”, and “fluorophore” asused herein refer to moieties that absorb light energy at a definedexcitation wavelength and emit light energy at a different wavelength.Examples of fluorescent labels include, but are not limited to: AlexaFluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, AlexaFluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, AlexaFluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 493/503, BODIPY 530/550,BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY630/650, BODIPY 650/665), Carboxyrhodamine 6G, carboxy-X-rhodamine(ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine dyes (Cy3,Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin,4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein, DM-NERF, Eosin, Erythrosin,Fluorescein, FAM, Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800),JOE, Lissamine rhodamine B, Marina Blue, Methoxycoumarin,Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, RhodamineGreen, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, YO-PRO-1, SYTOXGreen, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3,Fluo-4, fluoro-emerald, YoYo-1 ssDNA, YoYo-1 dsDNA, YoYo-1, SYTORNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX,Spectrum Green, NeuroTrace 500525, NBD-X, MitoTracker Green FM,LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), WEGFP(post-activation), FlASH-CCXXCC, Azami Green monomeric, Azami Green,green fluorescent protein (GFP), EGFP (Campbell Tsien 2003), EGFP(Patterson 2001), Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bexl, Doxorubicin, LumioGreen, and SuperGlo GFP.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

In the compounds of the invention, all of the atoms have sufficienthydrogen or non-hydrogen substituents to satisfy valence, or thecompound includes a pharmaceutically acceptable counterion, for examplein the case of a quaternary amine.

5.3.1 Grp94 Inhibitors of Formula (I)

In one aspect, the disclosure encompasses purine-scaffold compounds thatare substituted at the 8-position with a linker group bonded to an arylor heteroaryl group and are further substituted at the N-9 position.Such compounds are represented schematically in Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

(a) Y is —C(R^(Y))₂—, —S—, —NR—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—;

(c) Z² is —N— or —CR¹⁰—, wherein R¹⁰ is H or unsubstituted orsubstituted —(C₁-C₆)aliphatic;

(d) each of Z⁴, Z⁵, Z⁶, Z⁷ and Z⁸ are independently —C— or —N—, with theprovisos that at least one of Z⁴, Z⁶ and Z⁷ is —C— and no threeconsecutive Z⁴ through Z⁸ are N;

(e) X¹ is —H, -halo, —N(R)₂, —OR, —CN, or unsubstituted or substituted—(C₁-C₆)aliphatic;

(f) each of X², X³, X⁴, X⁵, and X⁶ are independently —H, -halo, —SR,—N(R)₂, —OR, —CN, —NO₂, —CN, —C(O)R, —C(O)₂R, —S(O)R, —S(O)₂R,—C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)SO₂R, —OC(O)N(R)₂,unsubstituted or substituted —(C₁-C₆)aliphatic, or an unsubstituted orsubstituted group selected from (5- or 6-membered)aryl, (5- or6-membered)arylalkyl, and (5- or 6-membered)heterocyclic aromatic orheterocyclic non-aromatic group; with the provisos that at least one ofX², X⁴ and X⁵ is —H and that X² is absent when Z⁴ is —N—, X³ is absentwhen Z⁵ is —N—, X⁴ is absent when Z⁶ is —N— and X⁵ is absent when Z⁷ is—N—;

(g) R¹ is —(C₁-C₆)aliphatic-N⁺—(R²)(R³)(R⁴), —(C₁-C₆)aliphatic-N—R³R⁴,—(C₁-C₆)aliphatic-C(═O)N—R³R⁴, —(C₁-C₆)aliphatic-R³R⁴,—(C₁-C₆)aliphatic-R²R³R⁴, —(C₁-C₆)aliphatic-N—CR²R³R⁴,—(C₁-C₆)aliphatic-C(halo)₃, —(C₁-C₆)aliphatic-alkenyl,—(C₁-C₆)aliphatic-alkynyl, —(C₁-C₆)aliphatic-(C₃-C₈)cycloalkyl,—(C₁-C₆)aliphatic-(C₃-C₈)heterocyclo, —(C₁-C₆)aliphatic-phenyl,—(C₁-C₆)aliphatic-(5 or 6-membered)heteroaryl, —(C₁-C₆)aliphatic-cyano,where the cycloalkyl, heterocyclo, heteroaryl, or phenyl isunsubstituted or substituted, with the proviso that when all of R²-R⁴are present the compound further comprises a pharmaceutically acceptablecounter ion;

(h) R² and R³ are independently hydrogen, —N(R)₂, —CH₂CH(OH)R⁴,—CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴, or unsubstituted or substituted—(C₁-C₆)aliphatic, or R³ and R⁴ form an unsubstituted or substituted 3-to 7-membered heterocyclic ring when taken together with the nitrogen towhich they are attached;

(i) R⁴ is hydrogen, halogen, or unsubstituted or substituted—(C₁-C₆)aliphatic;

(j) each R^(Y) is independently R, —OR, or halo;

(k) Z³ can be cyclized with X² to form a cyclic aryl, heteroaryl, alkylor heteroalkyl ring; and

(l) each R is independently hydrogen, unsubstituted C₁₋₆ aliphatic, orC₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂;

wherein each substituted group is substituted with one or more groupsselected from halo, —N(R)₂, —OR, —CN, oxo, unsubstituted C₁₋₆ aliphatic,or C₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂.

In some embodiments, a compound of formula (I) or pharmaceuticallyacceptable salt thereof is defined wherein:

(a) Y is —CH₂—, —S—, —NH—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—;

(c) Z² is —CH—, —N—, or —CR¹⁰—, wherein R¹⁰ is —(C₁-C₆)alkyl;

(d) each of Z⁴, Z⁵, Z⁶, Z⁷ and Z⁸ are independently —C— or —N—, with theprovisos that at least one of Z⁴, Z⁶ and Z⁷ is —C— and no threeconsecutive Z⁴ through Z⁸ are N;

(e) X¹ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, or—OCH₂(halo);

(f) each of X², X³, X⁴, and X⁵ are independently —H, -halo, —NH₂, —CN,—(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, —OCH₂(halo), or an unsubstituted orsubstituted (5- or 6-membered)aryl, heterocyclic aromatic ornon-aromatic group selected from pyridyl, furyl, thiophenyl, pyrrolyl,oxazolyl, imidazolyl, phenyl, benzyl, thiazolidinyl, thiadiazolyl,thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, triazinyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl,tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, ortetrahydrothiopyranyl, with the provisos that at least one of X², X⁴ andX⁵ is —H and that X² is absent when Z⁴ is —N—, X³ is absent when Z⁵ is—N—, X⁴ is absent when Z⁶ is —N— and X⁵ is absent when Z⁷ is —N—;

(g) X⁶ is —H when Z⁸ is —C— or absent when Z⁸ is —N—;

(h) R¹ is —(CH₂)_(m)—N⁺—(R²)(R³)(R⁴), —(CH₂)_(m)—N—R³R⁴,—(CH₂)_(m)—C(═O)N—R³R⁴, —(CH₂)_(m)—R³R⁴, —(CH₂)_(m)—C(halo)₃,—(CH₂)_(m)-alkenyl, —(CH₂)_(m)-alkenyl-CH₃, —(CH₂)_(m)-alkynyl,—(CH₂)_(m)-alkynyl-CH₃, —(CH₂)_(m)—(C₃-C₈)cycloalkyl,—(CH₂)_(m)—(C₃-C₈)heterocycloalkyl, —(CH₂)_(m)-phenyl, —(CH₂)_(m)-(5 or6-membered)heteroaryl, —(CH₂)_(m)-cyano, where m is 1, 2, 3, 4 or 5 andwhere the cycloalkyl, heterocycle or phenyl is unsubstituted orsubstituted with one or more X¹ groups, with the proviso that when allof R²-R⁴ are present the compound further comprises a pharmaceuticallyacceptable counter ion;

(i) R² and R³ are independently hydrogen, methyl, ethyl, ethenyl,ethynyl, propyl, butyl, pentyl, hexyl, isopropyl, c-propyl, t-butyl,isobutyl, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —CH₂C(halo)₃,—CH₂CH(halo)₂, —CH₂CH₂(halo), —NHCH₂C(halo)₃, —CH₂CH(halo)₂,—CH₂CH₂(halo), —CH₂OH, —CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH₂CH(CH₃)OH,—C(CH₃)₂CH₂OH, —CH₂CH(OH)R⁴, —CH₂SO₂NHR⁴, —CH₂SO₂NHR⁴ or R² and R³ forman unsubstituted or substituted aziridine, azetidine, pyrrolidine,piperazine, or piperidine ring when taken together with the nitrogen towhich they are attached;

(j) R⁴ is hydrogen, methyl, ethyl, isopropyl, t-butyl, isobutyl, or—C(halo)₃; and

(k) Z³ can be cyclized with X² to form a cyclic aryl, heteroaryl, alkylor heteroalkyl ring.

In one embodiment, Z¹, Z² and Z³ are —N—. In another embodiment, Z¹ andZ³ are —N— and Z² is —CH—. In another embodiment, Z¹ is —CH— and Z² andZ³ are —N—.

In another embodiment, Z⁴, Z⁵, Z⁶, Z⁷ and Z⁸ are —C—. In anotherembodiment, Z⁴ is —N— and Z⁵, Z⁶, Z⁷ and Z⁸ are —C—. In anotherembodiment, Z⁵ is —N— and Z⁴, Z⁶, Z⁷ and Z⁸ are —C—. In anotherembodiment, Z⁶ is —N— and Z⁴, Z⁵, Z⁷ and Z⁸ are —C—. In anotherembodiment, Z⁷ is —N— and Z⁴, Z⁵, Z⁶ and Z⁸ are —C—. In anotherembodiment, Z⁸ is —N— and Z⁴, Z⁵, Z⁶ and Z⁷ are —C—. In anotherembodiment, Z⁷ and Z⁴ are —N— and Z⁵, Z⁶ and Z⁸ are —C—. In anotherembodiment, Z⁵ and Z⁸ are —N— and Z⁴, Z⁶ and Z⁷ are —C—.

In another embodiment, Y is —S—, —CH₂—, or

In another embodiment, Y is S or

In another embodiment, Y is —S— or —CH₂—. In another embodiment, Y is—S— or —O—. In another embodiment, Y is —S—. In another embodiment, Y is—CH₂—. In another embodiment, Y is

In some embodiments, Y is —C(R^(Y))₂—, wherein each R^(Y) isindependently hydrogen, —OH, or halo.

In certain embodiments, R¹ is —(CH₂)_(m)—N—(R³)(R⁴). In one suchembodiment, R¹ is —(CH₂)₂—N—(R³)(R⁴). In another such embodiment, R¹ is—(CH₂)₃—N—(R³)(R⁴). In another such embodiment, R¹ is—(CH₂)₂—N—(R³)(R⁴), R³ is —H and R⁴ is isopropyl or isobutyl. In anothersuch embodiment, R¹ is —(CH₂)₃—N—(R³)(R⁴), R³ is —H and R⁴ is isopropylor isobutyl. In another such embodiment, R¹ is —(CH₂)₃—N—(R³)(R⁴), R³ is—H and R⁴ is isobutyl. In another such embodiment, R¹ is—(CH₂)₃—N—(R³)(R⁴), R³ is —H and R⁴ is isopropyl. It will be understood,that in these embodiments, the amine functionality may exist as a freebase or as an acid addition salt. Acid addition salts can be prepared byaddition of a suitable acid, as is well understood in the art. Inparticular embodiments, the acid addition salt may be a hydrochloridesalt, a phosphate salt, a sulfate salt, a lactate salt, a citrate salt,a succinate salt, a benzene sulfonic acid salt, a para-toluenesulfonicacid salt, or a fumaric acid-salt. In another embodiment, the acidaddition salt is a hydrochloride salt or a sulfate salt. In anotherembodiment, the acid addition salt is a hydrochloride salt. In anotherembodiment, the acid addition salt is a sulfate salt. In anotherembodiment, the acid addition salt is a phosphate salt. When prepared asan acid addition salt, the purine-scaffold inhibitors are rendered watersoluble. Solubility may be increased even further by production ofhigher order salts, particularly di-salts. For instance, in embodimentswhere Z¹ is —N—, the nitrogen is ionizable and can be converted to anacid addition salt under strongly acidic conditions (e.g., pH of lessthan 3). Accordingly, Grp94 inhibitors of the disclosure in which Z¹ is—N— and the R¹ group contains an amine functionality can be convertedinto di-salts. In certain embodiments, the Grp94 inhibitors of thedisclosure can be in the form of a di-HCl salt.

In some embodiments, R² and R³ are independently hydrogen, methyl,ethyl, ethenyl, ethynyl, propyl, butyl, pentyl, hexyl, isopropyl,c-propyl, t-butyl, isobutyl, —C(halo)₃, —CH(halo)₂, —CH₂(halo),—CH₂C(halo)₃, —CH₂CH(halo)₂, —CH₂CH₂(halo), —NHCH₂C(halo)₃,—CH₂CH(halo)₂, —CH₂CH₂(halo), —CH₂OH, —CH₂CH₂OH, —CH₂C(CH₃)₂OH,—CH₂CH(CH₃)OH, —C(CH₃)₂CH₂OH, —CH(CH₃)—CH₂OH, —CH(CH₃)CH(OH)R⁴,—CH₂CH(OH)R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴ or R² and R³ form anunsubstituted or substituted aziridine, azetidine, pyrrolidine,piperazine, or piperidine ring when taken together with the nitrogen towhich they are attached.

In some embodiments, R³ and R⁴ form an unsubstituted or substituted 3-to 7-membered heterocyclic ring when taken together with the nitrogen towhich they are attached. In some embodiments, R² and R³ form anunsubstituted or substituted 3- to 7-membered heterocyclic ring whentaken together with the nitrogen to which they are attached.

In certain embodiments, R¹ is —(CH₂)_(m)—CF₃. In one such embodiment, R¹is —(CH₂)₃—CF₃. In another such embodiment, R¹ is —(CH₂)₄—CF₃.

In some embodiments, R¹ is —(C₁-C₆)aliphatic-alkynyl. In someembodiments, R¹ is —(CH₂)₃CCH.

In some embodiments, R¹ is —(C₁-C₆)aliphatic-R³R⁴.

In some embodiments, R¹ is —(C₁-C₆)aliphatic-phenyl. In someembodiments, R¹ is —(C₁-C₆)aliphatic-heteroaryl. In some embodiments, R¹is —(C₁-C₆)aliphatic-heterocyclo.

In some embodiments, R¹ is —(CH₂)_(m)—NHR².

In certain embodiments, R¹ is —(CH₂)_(m)—C(═O)N—(R³)(R⁴). In one suchembodiment, R¹ is —(CH₂)₃—C(═O)NH₂. In another such embodiment, R¹ is(CH₂)₄—C(═O)NH₂. In another such embodiment, R¹ is (CH₂)₅—C(═O)NH₂.

In another embodiment, X¹ is —H. In another embodiment, X¹ is a halogenatom. In another embodiment, X¹ is —F. In another embodiment, X¹ is —Cl.

In another embodiment, X² is a halogen atom, —OCH₃, or —OCF₃ and X³, X⁴,X⁵ and X⁶ are —H. In another embodiment X² is —Cl and X³, X⁴, X⁵ and X⁶are —H. In another embodiment X² is —OCH₃ and X³, X⁴, X⁵ and X⁶ are —H.In another embodiment X² is —OCF₃ and X³, X⁴, X⁵ and X⁶ are —H.

In another embodiment, X⁴ is a halogen atom and X², X³, X⁵ and X⁶ are—H. In another embodiment X⁴ is —Cl and X², X³, X⁵ and X⁶ are —H. Inanother embodiment X⁴ is —OCH₃ and X², X³, X⁵ and X⁶ are —H. In anotherembodiment X⁴ is —OCF₃ and X², X³, X⁵ and X⁶ are —H.

In some embodiments X², X³, and X⁵ are halogen and X⁴ and X⁶ arehydrogen. In some embodiments X², X³, and X⁴ are halogen and X⁵ and X⁶are hydrogen. In some embodiments X², X³, and X⁵ are halogen and X⁴ andX⁶ are hydrogen. In some embodiments X³, X⁴, and X⁵ are halogen and X²and X⁶ are hydrogen.

In some embodiments, X², X⁴, and X⁶ are methyl and X³ and X⁵ arehydrogen.

In certain embodiments, Z⁴ and Z⁶ are —C—, X² and X⁴ are independentlyselected from —H, -halo, —(C₁-C₃)alkyl and —O(C₁-C₃)alkyl and Z⁵, Z⁷ andZ⁸ are either an unsubstituted carbon or a nitrogen atom. In one suchembodiment, at least one of X² and X⁴ are -halo. In another suchembodiment, both X² and X⁴ are —Cl. In another such embodiment, at leastone of X² and X⁴ are alkyl groups. In another such embodiment, both X²and X⁴ are —CH₃. In another such embodiment, at least one of X² and X⁴are —OCH₃. In another such embodiment, at least one of X² and X⁴ are—CF₃.

In certain embodiments, Z⁴ and Z⁷ are —C—, X² and X⁵ are independentlyselected from —H, -halo, —(C₁-C₃)alkyl and —O(C₁-C₃)alkyl and Z⁵, Z⁶ andZ⁸ are either an unsubstituted carbon or a nitrogen atom. In one suchembodiment, at least one of X² and X⁵ are halogen atoms. In another suchembodiment, both X² and X⁵ are —Cl. In another such embodiment, at leastone of X² and X⁴ are alkyl groups. In another such embodiment, both X²and X⁵ are —CH₃. In another such embodiment, at least one of X² and X⁴are —CF₃.

In certain embodiments, Z⁵ and Z⁷ are —C—, X³ and X⁵ are independentlyselected from —H, -halo, —(C₁-C₃)alkyl, —O(C₁-C₃)alkyl, unsubstituted orsubstituted —(C₁-C₆)aliphatic, or unsubstituted or substituted phenyl,and Z⁴, Z⁶ and Z⁸ are either an unsubstituted carbon or a nitrogen atom.In one such embodiment, at least one of X³ and X⁵ are halogen atoms. Inanother such embodiment, both X³ and X⁵ are —Cl. In another suchembodiment, at least one of X³ and X⁵ are alkyl groups. In another suchembodiment, both X³ and X⁵ are —CH₃. In another such embodiment, atleast one of X³ and X⁵ are —CF₃.

In some embodiments, the Grp94 inhibitors of Formula (I) are of Formula(Ia):

or a pharmaceutically acceptable salt thereof, wherein each of X¹, Z²,R¹, Y, X³, and X⁵ is as defined above and described in classes andsubclasses herein, both singly and in combination.

In some embodiments, the Grp94 inhibitors of Formula (I) are of Formula(Ib):

or a pharmaceutically acceptable salt thereof, wherein R¹ is as definedabove where i) the —(C₁-C₆)aliphatic group attached to the ring nitrogenis —(CH₂)₃— or ii) m is 3; and each of X¹, Z², Y, X², X³, X⁴, X⁵ and X⁶is as defined above and described in classes and subclasses herein, bothsingly and in combination.

In some embodiments, the Grp94 inhibitors of Formula (I) have one of theFormula of Table 2, wherein each substituent is as defined above anddescribed in classes and subclasses herein, both singly and incombination.

TABLE 2 Formula Compound IA

IB

IC

ID

IE

IF

IG

IH

II

IJ

IK

IL

IM

IN

IO

IP

IQ

IR

IS

IT

IU

IV

IW

IX

IY

IZ

IAA

IAB

IAC

IAD

IAE

IAF

IAG

IAH

IAI

IAJ

IAK

IAL

IAM

IAN

IAO

IAP

IAQ

IAR

IAS

IAT

IAU

IAV

IAW

IAX

IAY

IAZ

IBA

IBB

IBC

IBD

IBE

IBF

IBG

IBH

IBI

IBJ

IBK

IBL

IBM

IBN

IBO

IBP

IBQ

IBR

IBS

IBT

IBU

IBV

IBW

IBX

IBY

IBZ

ICA

ICB

ICC

ICD

ICE

ICF

ICC

ICD

ICE

ICF

ICG

ICH

ICI

ICJ

ICK

ICL

ICM

ICN

ICO

ICP

ICQ

ICR

ICS

ICT

ICU

ICV

ICW

ICX

ICY

ICZ

IDA

IDB

IDC

IDD

IDE

IDF

IDG

IDH

Illustrative compounds of Formula (I) are listed below in Tables 2A, 2B,2C, 2D and 3.

TABLE 2A

and pharmaceutically acceptable salts thereof, where: m R⁴ Y X² X³ X⁴ X⁵2 CH(CH₃)₂ S H Cl H H 2 CH₂CH₂OH S H H H Cl 2 CH₂CH(CH₃)OH S H H Cl H 2CH(CH₃)CH₂OH S Cl H H H 2 CH(CH₃)CH(CH₃)OH S H Br H H 2 C(CH₃)₂CH₂OH S HH H Br 2 CH₂C(CH₃)₂OH S H H Br H 2 CH₂CHF₂ S Br H H H 2 CH₂CF₃ S H I H H2 CH₂CH(CH₃)₂ S H H H I 2 CH₂C(CH₃)₃ S H H I H 2 H₂C—C≡N S I H H H 3H₂C—C≡CH S H CH₃ H H 3 CH(CH₃)₂ S H H H CH₃ 3 CH₂CH₂OH S H H CH₃ H 3CH₂CH(CH₃)OH S CH₃ H H H 3 CH(CH₃)CH₂OH S H C₂H₅ H H 3 CH(CH₃)CH(CH₃)OHS H H H C₂H₅ 3 C(CH₃)₂CH₂OH S H H C₂H₅ H 3 CH₂C(CH₃)₂OH S C₂H₅ H H H 3CH₂CHF₂ S H i- H H C₃H₇ 3 CH₂CF₃ S H H H i- C₃H₇ 3 CH₂CH(CH₃)₂ S H H i-H C₃H₇ 3 CH₂C(CH₃)₃ S i- H H H C₃H₇ 3 H₂C—C≡N O H Cl H H 2 H₂C—C≡CH O HH H Cl 2 CH(CH₃)₂ O H H Cl H 2 CH₂CH₂OH O Cl H H H 2 CH₂CH(CH₃)OH O H BrH H 2 CH(CH₃)CH₂OH O H H H Br 2 CH(CH₃)CH(CH₃)OH O H H Br H 2C(CH₃)₂CH₂OH O Br H H H 2 CH₂C(CH₃)₂OH O H I H H 2 CH₂CHF₂ O H H H I 2CH₂CF₃ O H H I H 2 CH₂CH(CH₃)₂ O I H H H 3 CH₂C(CH₃)₃ O H CH₃ H H 3H₂C—C≡N O H H H CH₃ 3 H₂C—C≡CH O H H CH₃ H 3 CH(CH₃)₂ O CH₃ H H H 3CH₂CH₂OH O H C₂H₅ H H 3 CH₂CH(CH₃)OH O H H H C₂H₅ 3 CH(CH₃)CH₂OH O H HC₂H₅ H 3 CH(CH₃)CH(CH₃)OH O C₂H₅ H H H 3 C(CH₃)₂CH₂OH O H i- H H C₃H₇ 3CH₂C(CH₃)₂OH O H H H i- C₃H₇ 3 CH₂CHF₂ O H H i- H C₃H₇ 3 CH₂CF₃ O i- H HH C₃H₇ 2 CH₂CH(CH₃)₂ S═O H Cl H H 2 CH₂C(CH₃)₃ S═O H H H Cl 2 H₂C—C≡NS═O H H Cl H 2 H₂C—C≡CH S═O Cl H H H 2 CH(CH₃)₂ S═O H Br H H 2 CH₂CH₂OHS═O H H H Br 2 CH₂CH(CH₃)OH S═O H H Br H 2 CH(CH₃)CH₂OH S═O Br H H H 2CH(CH₃)CH(CH₃)OH S═O H I H H 2 C(CH₃)₂CH₂OH S═O H H H I 2 CH₂C(CH₃)₂OHS═O H H I H 2 CH₂CHF₂ S═O I H H H 3 CH₂CF₃ S═O H CH₃ H H 3 CH₂CH(CH₃)₂S═O H H H CH₃ 3 CH₂C(CH₃)₃ S═O H H CH₃ H 3 H₂C—C≡N S═O CH₃ H H H 3H₂C—C≡CH S═O H C₂H₅ H H 3 CH(CH₃)₂ S═O H H H C₂H₅ 3 CH₂CH₂OH S═O H HC₂H₅ H 3 CH₂CH(CH₃)OH S═O C₂H₅ H H H 3 CH(CH₃)CH₂OH S═O H i- H H C₃H₇ 3CH(CH₃)₂ S═O H H H i- C₃H₇ 3 CH₂CH₂OH S═O H H i- H C₃H₇ 3 CH₂CH(CH₃)OHS═O i- H H H C₃H₇ 2 CH(CH₃)CH₂OH O═S═O H Cl H H 2 CH(CH₃)CH(CH₃)OH O═S═OH H H Cl 2 C(CH₃)₂CH₂OH O═S═O H H Cl H 2 CH₂C(CH₃)₂OH O═S═O Cl H H H 2CH₂CHF₂ O═S═O H Br H H 2 CH₂CF₃ O═S═O H H H Br 2 CH₂CH(CH₃)₂ O═S═O H HBr H 2 CH₂C(CH₃)₃ O═S═O Br H H H 2 H₂C—C≡N O═S═O H I H H 2 H₂C—C≡CHO═S═O H H H I 2 CH(CH₃)₂ O═S═O H H I H 2 CH₂CH₂OH O═S═O I H H H 3CH₂CH(CH₃)OH O═S═O H CH₃ H H 3 CH(CH₃)CH₂OH O═S═O H H H CH₃ 3CH(CH₃)CH(CH₃)OH O═S═O H H CH₃ H 3 C(CH₃)₂CH₂OH O═S═O CH₃ H H H 3CH₂C(CH₃)₂OH O═S═O H C₂H₅ H H 3 CH₂CHF₂ O═S═O H H H C₂H₅ 3 CH₂CF₃ O═S═OH H C₂H₅ H 3 CH₂CH(CH₃)₂ O═S═O C₂H₅ H H H 3 CH₂C(CH₃)₃ O═S═O H i- H HC₃H₇ 3 H₂C—C≡N O═S═O H H H i- C₃H₇ 3 H₂C—C≡CH O═S═O H H i- H C₃H₇ 3CH(CH₃)₂ O═S═O i- H H H C₃H₇ 2 CH₂CH₂OH NH H Cl H H 2 CH₂CH(CH₃)OH NH HH H Cl 2 CH(CH₃)CH₂OH NH H H Cl H 2 CH(CH₃)CH(CH₃)OH NH Cl H H H 2C(CH₃)₂CH₂OH NH H Br H H 2 CH₂C(CH₃)₂OH NH H H H Br 2 CH₂CHF₂ NH H H BrH 2 CH₂CF₃ NH Br H H H 2 CH₂CH(CH₃)₂ NH H I H H 2 CH₂C(CH₃)₃ NH H H H I2 H₂C—C≡N NH H H I H 2 H₂C—C≡CH NH I H H H 3 CH(CH₃)₂ NH H CH₃ H H 3CH₂CH₂OH NH H H H CH₃ 3 CH₂CH(CH₃)OH NH H H CH₃ H 3 CH(CH₃)CH₂OH NH CH₃H H H 3 CH(CH₃)CH(CH₃)OH NH H C₂H₅ H H 3 C(CH₃)₂CH₂OH NH H H H C₂H₅ 3CH₂C(CH₃)₂OH NH H H C₂H₅ H 3 CH₂CHF₂ NH C₂H₅ H H H 3 CH₂CF₃ NH H i- H HC₃H₇ 3 CH₂CH(CH₃)₂ NH H H H i- C₃H₇ 3 CH₂C(CH₃)₃ NH H H i- H C₃H₇ 3H₂C—C≡N NH i- H H H C₃H₇ 2 H₂C—C≡CH C═O H Cl H H 2 CH(CH₃)₂ C═O H H H Cl2 CH₂CH₂OH C═O H H Cl H 2 CH₂CH(CH₃)OH C═O Cl H H H 2 CH(CH₃)CH₂OH C═O HBr H H 2 CH(CH₃)CH(CH₃)OH C═O H H H Br 2 C(CH₃)₂CH₂OH C═O H H Br H 2CH₂C(CH₃)₂OH C═O Br H H H 2 CH₂CHF₂ C═O H I H H 2 CH₂CF₃ C═O H H H I 2CH₂CH(CH₃)₂ C═O H H I H 2 CH₂C(CH₃)₃ C═O I H H H 3 H₂C—C≡N C═O H CH₃ H H3 H₂C—C≡CH C═O H H H CH₃ 3 CH(CH₃)₂ C═O H H CH₃ H 3 CH₂CH₂OH C═O CH₃ H HH 3 CH₂CH(CH₃)OH C═O H C₂H₅ H H 3 CH(CH₃)CH₂OH C═O H H H C₂H₅ 3CH(CH₃)CH(CH₃)OH C═O H H C₂H₅ H 3 C(CH₃)₂CH₂OH C═O C₂H₅ H H H 3CH₂C(CH₃)₂OH C═O H i- H H C₃H₇ 3 CH₂CHF₂ C═O H H H i- C₃H₇ 3 CH₂CF₃ C═OH H i- H C₃H₇ 3 CH₂CH(CH₃)₂ C═O i- H H H C₃H₇ 2 CH₂C(CH₃)₃ C═S H Cl H H2 H₂C—C≡N C═S H H H Cl 2 H₂C—C≡CH C═S H H Cl H 2 CH(CH₃)₂ C═S Cl H H H 2CH₂CH₂OH C═S H Br H H 2 CH₂CH(CH₃)OH C═S H H H Br 2 CH(CH₃)CH₂OH C═S H HBr H 2 CH(CH₃)CH(CH₃)OH C═S Br H H H 2 C(CH₃)₂CH₂OH C═S H I H H 2CH₂C(CH₃)₂OH C═S H H H I 2 CH₂CHF₂ C═S H H I H 2 CH₂CF₃ C═S I H H H 3CH₂CH(CH₃)₂ C═S H CH₃ H H 3 CH₂C(CH₃)₃ C═S H H H CH₃ 3 H₂C—C≡N C═S H HCH₃ H 3 H₂C—C≡CH C═S CH₃ H H H 3 CH(CH₃)₂ C═S H C₂H₅ H H 3 CH₂CH₂OH C═SH H H C₂H₅ 3 CH₂CH(CH₃)OH C═S H H C₂H₅ H 3 CH(CH₃)CH₂OH C═S C₂H₅ H H H 3CH(CH₃)CH(CH₃)OH C═S H i- H H C₃H₇ 3 C(CH₃)₂CH₂OH C═S H H H i- C₃H₇ 3CH₂C(CH₃)₂OH C═S H H i- H C₃H₇ 3 CH₂CHF₂ C═S i- H H H C₃H₇ 2 CH₂CF₃ CH₂H Cl H H 2 CH₂CH(CH₃)₂ CH₂ H H H Cl 2 CH₂C(CH₃)₃ CH₂ H H Cl H 2 H₂C—C≡NCH₂ Cl H H H 2 H₂C—C≡CH CH₂ H Br H H 2 CH(CH₃)₂ CH₂ H H H Br 2 CH₂CH₂OHCH₂ H H Br H 2 CH₂CH(CH₃)OH CH₂ Br H H H 2 CH(CH₃)CH₂OH CH₂ H I H H 2CH(CH₃)CH(CH₃)OH CH₂ H H H I 2 C(CH₃)₂CH₂OH CH₂ H H I H 2 CH₂C(CH₃)₂OHCH₂ I H H H 3 CH₂CHF₂ CH₂ H CH₃ H H 3 CH₂CF₃ CH₂ H H H CH₃ 3 CH₂CH(CH₃)₂CH₂ H H CH₃ H 3 CH₂C(CH₃)₃ CH₂ CH₃ H H H 3 H₂C—C≡N CH₂ H C₂H₅ H H 3H₂C—C≡CH CH₂ H H H C₂H₅ 3 CH(CH₃)₂ CH₂ H H C₂H₅ H 3 CH₂CH₂OH CH₂ C₂H₅ HH H 3 CH₂CH(CH₃)OH CH₂ H i- H H C₃H₇ 3 CH(CH₃)CH₂OH CH₂ H H H i- C₃H₇ 3CH(CH₃)CH(CH₃)OH CH₂ H H i- H C₃H₇ 3 C(CH₃)₂CH₂OH CH₂ i- H H H C₃H₇ 2CH₂C(CH₃)₂OH CH—OH H Cl H H 2 CH₂CHF₂ CH—OH H H H Cl 2 CH₂CF₃ CH—OH H HCl H 2 CH₂CH(CH₃)₂ CH—OH Cl H H H 2 CH₂C(CH₃)₃ CH—OH H Br H H 2 H₂C—C≡NCH—OH H H H Br 2 H₂C—C≡CH CH—OH H H Br H 2 CH(CH₃)₂ CH—OH Br H H H 2CH₂CH₂OH CH—OH H I H H 2 CH₂CH(CH₃)OH CH—OH H H H I 2 CH(CH₃)CH₂OH CH—OHH H I H 2 CH(CH₃)CH(CH₃)OH CH—OH I H H H 3 C(CH₃)₂CH₂OH CH—OH H CH₃ H H3 CH₂C(CH₃)₂OH CH—OH H H H CH₃ 3 CH₂CHF₂ CH—OH H H CH₃ H 3 CH₂CF₃ CH—OHCH₃ H H H 3 CH₂CH(CH₃)₂ CH—OH H C₂H₅ H H 3 CH₂C(CH₃)₃ CH—OH H H H C₂H₅ 3H₂C—C≡N CH—OH H H C₂H₅ H 3 H₂C—C≡CH CH—OH C₂H₅ H H H 3 CH(CH₃)₂ CH—OH Hi- H H C₃H₇ 3 CH₂CH₂OH CH—OH H H H i- C₃H₇ 3 CH₂CH(CH₃)OH CH—OH H H i- HC₃H₇ 3 CH(CH₃)CH₂OH CH—OH i- H H H C₃H₇ 2 CH(CH₃)CH(CH₃)OH CH—F H Cl H H2 C(CH₃)₂CH₂OH CH—F H H H Cl 2 CH₂C(CH₃)₂OH CH—F H H Cl H 2 CH₂CHF₂ CH—FCl H H H 2 CH₂CF₃ CH—F H Br H H 2 CH₂CH(CH₃)₂ CH—F H H H Br 2 CH₂C(CH₃)₃CH—F H H Br H 2 H₂C—C≡N CH—F Br H H H 2 H₂C—C≡CH CH—F H I H H 2 CH(CH₃)₂CH—F H H H I 2 CH₂CH₂OH CH—F H H I H 2 CH₂CH(CH₃)OH CH—F I H H H 3CH(CH₃)CH₂OH CH—F H CH₃ H H 3 CH(CH₃)CH(CH₃)OH CH—F H H H CH₃ 3C(CH₃)₂CH₂OH CH—F H H CH₃ H 3 CH₂C(CH₃)₂OH CH—F CH₃ H H H 3 CH₂CHF₂ CH—FH C₂H₅ H H 3 CH₂CF₃ CH—F H H H C₂H₅ 3 CH₂CH(CH₃)₂ CH—F H H C₂H₅ H 3CH₂C(CH₃)₃ CH—F C₂H₅ H H H 3 H₂C—C≡N CH—F H i- H H C₃H₇ 3 H₂C—C≡CH CH—FH H H i- C₃H₇ 3 CH(CH₃)₂ CH—F H H i- H C₃H₇ 3 CH₂CH₂OH CH—F i- H H HC₃H₇ 2 CH₂CH(CH₃)OH S H Cl H Cl 2 CH(CH₃)CH₂OH S Cl H Cl H 2CH(CH₃)CH(CH₃)OH S Cl H H Cl 2 C(CH₃)₂CH₂OH S H Br H Br 2 CH₂C(CH₃)₂OH SBr H Br H 2 CH₂CHF₂ S Br H H Br 2 CH₂CF₃ S H I H I 2 CH₂CH(CH₃)₂ S I H IH 2 CH₂C(CH₃)₃ S I H H I 2 H₂C—C≡N S H CH₃ H CH₃ 2 H₂C—C≡CH S CH H CH₃ H2 CH(CH₃)₂ S CH H H CH₃ 3 CH₂CH₂OH S H C₂H₅ H C₂H₅ 3 CH₂CH(CH₃)OH S C₂H₅H C₂H₅ H 3 CH(CH₃)CH₂OH S C₂H₅ H H C₂H₅ 3 CH(CH₃)CH(CH₃)OH S H i- H i-C₃H₇ C₃H₇ 3 C(CH₃)₂CH₂OH S i- H i- H C₃H₇ C₃H₇ 3 CH₂C(CH₃)₂OH S i- H Hi- C₃H₇ C₃H₇ 3 CH₂CHF₂ O H Cl H Cl 3 CH₂CF₃ O Cl H Cl H 3 CH₂CH(CH₃)₂ OCl H H Cl 3 CH₂C(CH₃)₃ O H Br H Br 3 H₂C—C≡N O Br H Br H 3 H₂C—C≡CH O BrH H Br 2 CH(CH₃)₂ O H I H I 2 CH₂CH₂OH O I H I H 2 CH₂CH(CH₃)OH O I H HI 2 CH(CH₃)CH₂OH O H CH₃ H CH₃ 2 CH(CH₃)CH(CH₃)OH O CH₃ H CH₃ H 2C(CH₃)₂CH₂OH O CH₃ H H CH₃ 2 CH₂C(CH₃)₂OH O H C₂H₅ H C₂H₅ 2 CH₂CHF₂ OC₂H₅ H C₂H₅ H 2 CH₂CF₃ O C₂H₅ H H C₂H₅ 2 CH₂CH(CH₃)₂ O H i- H i- C₃H₇C₃H₇ 2 CH₂C(CH₃)3 O i- H i- H C₃H₇ C₃H₇ 2 H₂C—C≡N O i- H H i- C₃H₇ C₃H₇3 H₂C—C≡CH S═O H Cl H Cl 3 CH(CH₃)₂ S═O Cl H Cl H 3 CH₂CH₂OH S═O Cl H HCl 3 CH₂CH(CH₃)OH S═O H Br H Br 3 CH(CH₃)CH₂OH S═O Br H Br H 3CH(CH₃)CH(CH₃)OH S═O Br H H Br 3 C(CH₃)₃CH₂OH S═O H I H I 3 CH₂C(CH₃)₂OHS═O I H I H 3 CH₂CHF₂ S═O I H H I 3 CH₂CF₃ S═O H CH₃ H CH₃ 3 CH₂CH(CH₃)₂S═O CH₃ H CH₃ H 3 CH₂C(CH₃)₃ S═O CH₃ H H CH₃ 2 H₂C—C≡N S═O H C₂H₅ H C₂H₅2 H₂C—C≡CH S═O C₂H₅ H C₂H₅ H 2 CH(CH₃)₂ S═O C₂H₅ H H C₂H₅ 2 CH₂CH₂OH S═OH i- H i- C₃H₇ C₃H₇ 2 CH₂CH(CH₃)OH S═O i- H i- H C₃H₇ C₃H₇ 2CH(CH₃)CH₂OH S═O i- H H i- C₃H₇ C₃H₇ 2 CH(CH₃)CH(CH₃)OH O═S═O H Cl H Cl2 C(CH₃)₂CH₂OH O═S═O Cl H Cl H 2 CH₂C(CH₃)₂OH O═S═O Cl H H Cl 2 CH₂CHF₂O═S═O H Br H Br 2 CH₂CF₃ O═S═O Br H Br H 2 CH₂CH(CH₃)₂ O═S═O Br H H Br 3CH₂C(CH₃)₃ O═S═O H I H I 3 H₂C—C≡N O═S═O I H I H 3 H₂C—C≡CH O═S═O I H HI 3 CH(CH₃)₂ O═S═O H CH₃ H CH₃ 3 CH₂CH₂OH O═S═O CH₃ H CH₃ H 3CH₂CH(CH₃)OH O═S═O CH₃ H H CH₃ 3 CH(CH₃)CH₂OH O═S═O H C₂H₅ H C₂H₅ 3CH(CH₃)CH(CH₃)OH O═S═O C₂H₅ H C₂H₅ H 3 C(CH₃)₂CH₂OH O═S═O C₂H₅ H H C₂H₅3 CH₂C(CH₃)₂OH O═S═O H i- H i- C₃H₇ C₃H₇ 3 CH₂CHF₂ O═S═O i- H i- H C₃H₇C₃H₇ 3 CH₂CF₃ O═S═O i- H H i- C₃H₇ C₃H₇ 2 CH₂CH(CH₃)₂ NH H Cl H Cl 2CH₂C(CH₃)₃ NH Cl H Cl H 2 H₂C—C≡N NH Cl H H Cl 2 H₂C—C≡CH NH H Br H Br 2CH(CH₃)₂ NH Br H Br H 2 CH₂CH₂OH NH Br H H Br 2 CH₂CH(CH₃)OH NH H I H I2 CH(CH₃)CH₂OH NH I H I H 2 CH(CH₃)CH(CH₃)OH NH I H H I 2 C(CH₃)₂CH₂OHNH H CH₃ H CH₃ 2 CH₂C(CH₃)₂OH NH CH₃ H CH₃ H 2 CH₂CHF₂ NH CH₃ H H CH₃ 3CH₂CF₃ NH H C₂H₅ H C₂H₅ 3 CH₂CH(CH₃)₂ NH C₂H₅ H C₂H₅ H 3 CH₂C(CH₃)₃ NHC₂H₅ H H C₂H₅ 3 H₂C—C≡N NH H i- H i- C₃H₇ C₃H₇ 3 H₂C—C≡CH NH i- H i- HC₃H₇ C₃H₇ 3 CH(CH₃)₂ NH i- H H i- C₃H₇ C₃H₇ 3 CH₂CH₂OH C═O H Cl H Cl 3CH₂CH(CH₃)OH C═O Cl H Cl H 3 CH(CH₃)CH₂OH C═O Cl H H Cl 3CH(CH₃)CH(CH₃)OH C═O H Br H Br 3 C(CH₃)₂CH₂OH C═O Br H Br H 3CH₂C(CH₃)₂OH C═O Br H H Br 3 CH₂CHF₂ C═O H I H I 2 CH₂CF₃ C═O I H I H 2CH₂CH(CH₃)₂ C═O I H H I 2 CH₂C(CH₃)₃ C═O H CH₃ H CH₃ 2 H₂C—C≡N C═O CH₃ HCH₃ H 2 H₂C—C≡CH C═O CH₃ H H CH₃ 2 CH(CH₃)₂ C═O H C₂H₅ H C₂H₅ 2 CH₂CH₂OHC═O C₂H₅ H C₂H₅ H 2 CH₂CH(CH₃)OH C═O C₂H₅ H H C₂H₅ 2 CH(CH₃)CH₂OH C═O Hi- H i- C₃H₇ C₃H₇ 2 CH(CH₃)CH(CH₃)OH C═O i- H i- H C₃H₇ C₃H₇ 2C(CH₃)₂CH₂OH C═O i- H H i- C₃H₇ C₃H₇ 3 CH₂C(CH₃)₂OH C═S H Cl H Cl 3CH₂CHF₂ C═S Cl H Cl H 3 CH₂CF₃ C═S Cl H H Cl 3 CH₂CH(CH₃)₂ C═S H Br H Br3 CH₂C(CH₃)₃ C═S Br H Br H 3 H₂C—C≡N C═S Br H H Br 3 H₂C—C≡CH C═S H I HI 3 CH(CH₃)₂ C═S I H I H 3 CH₂CH₂OH C═S I H H I 3 CH₂CH(CH₃)OH C═S H CH₃H CH₃ 2 CH(CH₃)CH₂OH C═S CH₃ H CH₃ H 2 CH(CH₃)CH(CH₃)OH C═S CH H H CH₃ 2C(CH₃)₂CH₂OH C═S H C₂H₅ H C₂H₅ 2 CH₂C(CH₃)₂OH C═S C₂H₅ H C₂H₅ H 2CH₂CHF₂ C═S C₂H₅ H H C₂H₅ 2 CH₂CF₃ C═S H i- H i- C₃H₇ C₃H₇ 2 CH₂CH(CH₃)₂C═S i- H i- H C₃H₇ C₃H₇ 2 CH₂C(CH₃)₃ C═S i- H H i- C₃H₇ C₃H₇ 3 H₂C—C≡NCH₂ H Cl H Cl 3 H₂C—C≡CH CH₂ Cl H Cl H 3 CH(CH₃)₂ CH₂ Cl H H Cl 3CH₂CH₂OH CH₂ H Br H Br 3 CH₂CH(CH₃)OH CH₂ Br H Br H 3 CH(CH₃)CH₂OH CH₂Br H H Br 2 CH(CH₃)CH(CH₃)OH CH₂ H I H I 2 C(CH₃)₂CH₂OH CH₂ I H I H 2CH₂C(CH₃)₂OH CH₂ I H H I 2 CH₂CHF₂ CH₂ H CH₃ H CH₃ 2 CH₂CF₃ CH₂ CH₃ HCH₃ H 2 CH₂CH(CH₃)₂ CH₂ CH₃ H H CH₃ 3 CH₂C(CH₃)₃ CH₂ H C₂H₅ H C₂H₅ 3H₂C—C≡N CH₂ C₂H₅ H C₂H₅ H 3 H₂C—C≡CH CH₂ C₂H₅ H H C₂H₅ 3 CH(CH₃)₂ CH₂ Hi- H i- C₃H₇ C₃H₇ 3 CH₂CH₂OH CH₂ i- H i- H C₃H₇ C₃H₇ 3 CH₂CH(CH₃)OH CH₂i- H H i- C₃H₇ C₃H₇ 2 CH(CH₃)CH₂OH CH—OH H Cl H Cl 2 CH(CH₃)CH(CH₃)OHCH—OH Cl H Cl H 2 C(CH₃)₂CH₂OH CH—OH Cl H H Cl 2 CH₂C(CH₃)₂OH CH—OH H BrH Br 2 CH₂CHF₂ CH—OH Br H Br H 2 CH₂CF₃ CH—OH Br H H Br 3 CH₂CH(CH₃)2CH—OH H I H I 3 CH₂C(CH₃)₃ CH—OH I H I H 3 H₂C—C≡N CH—OH I H H I 3H₂C—C≡CH CH—OH H CH₃ H CH₃ 3 CH(CH₃)₂ CH—OH CH₃ H CH₃ H 3 CH₂CH₂OH CH—OHCH₃ H H CH₃ 2 CH₂CH(CH₃)OH CH—OH H C₂H₅ H C₂H₅ 2 CH(CH₃)CH₂OH CH—OH C₂H₅H C₂H₅ H 2 CH(CH₃)CH(CH₃)OH CH—OH C₂H₅ H H C₂H₅ 2 C(CH₃)₂CH₂OH CH—OH Hi- H i- C₃H₇ C₃H₇ 2 CH₂C(CH₃)₂OH CH—OH i- H i- H C₃H₇ C₃H₇ 2 CH₂CHF₂CH—OH i- H H i- C₃H₇ C₃H₇ 3 CH₂CF₃ CH—F H Cl H Cl 3 CH₂CH(CH₃)₂ CH—F ClH Cl H 3 CH₂C(CH₃)₃ CH—F Cl H H Cl 3 H₂C—C≡N CH—F H Br H Br 3 H₂C—C≡CHCH—F Br H Br H 3 CH(CH₃)₂ CH—F Br H H Br 2 CH₂CH₂OH CH—F H I H I 2CH₂CH(CH₃)OH CH—F I H I H 2 CH(CH₃)CH₂OH CH—F I H H I 2 CH(CH₃)CH(CH₃)OHCH—F H CH₃ H CH₃ 2 C(CH₃)₂CH₂OH CH—F CH₃ H CH₃ H 2 CH₂C(CH₃)₂OH CH—F CH₃H H CH₃ 3 CH₂CHF₂ CH—F H C₂H₅ H C₂H₅ 3 CH₂CF₃ CH—F C₂H₅ H C₂H₅ H 3CH₂CH(CH₃)₂ CH—F C₂H₅ H H C₂H₅ 3 CH₂C(CH₃)₃ CH—F H i- H i- C₃H₇ C₃H₇ 3H₂C—C≡N CH—F i- H i- H C₃H₇ C₃H₇ 3 H₂C—C≡CH CH—F i- H H i- C₃H₇ C₃H₇ 2CH(CH₃)₂ S H Cl Cl Cl 2 CH₂CH₂OH S Cl Cl H Cl 2 CH₂CH(CH₃)OH S H Br BrBr 2 CH(CH₃)CH₂OH S Br Br H Br 2 CH(CH₃)CH(CH₃)OH S H I I I 2C(CH₃)₂CH₂OH S I I H I 3 CH₂C(CH₃)₂OH S H CH₃ CH₃ CH₃ 3 CH₂CHF₂ S CH₃CH₃ H CH₃ 3 CH₂CF₃ S H C₂H₅ C₂H₅ C₂H₅ 3 CH₂CH(CH₃)₂ S C₂H₅ C₂H₅ H C₂H₅ 3CH₂C(CH₃)₃ S H i- i- i- C₃H₇ C₃H₇ C₃H₇ 3 H₂C—C≡N S i- i- H i- C₃H₇ C₃H₇C₃H₇ 2 H₂C—C≡CH O H Cl Cl Cl 2 CH(CH₃)₂ O Cl Cl H Cl 2 CH₂CH₂OH O H BrBr Br 2 CH₂CH(CH₃)OH O Br Br H Br 2 CH(CH₃)CH₂OH O H I I I 2CH(CH₃)CH(CH₃)OH O I I H I 3 C(CH₃)₂CH₂OH O H CH₃ CH₃ CH₃ 3 CH₂C(CH₃)₂OHO CH₃ CH₃ H CH₃ 3 CH₂CHF₂ O H C₂H₅ C₂H₅ C₂H₅ 3 CH₂CF₃ O C₂H₅ C₂H₅ H C₂H₅3 CH₂CH(CH₃)₂ O H i- i- i- C₃H₇ C₃H₇ C₃H₇ 3 CH₂C(CH₃)₃ O i- i- H i- C₃H₇C₃H₇ C₃H₇ 2 H₂C—C≡N S═O H Cl Cl Cl 2 H₂C—C≡CH S═O Cl Cl H Cl 2 CH(CH₃)₂S═O H Br Br Br 2 CH₂CH₂OH S═O Br Br H Br 2 CH₂CH(CH₃)OH S═O H I I I 2CH(CH₃)CH₂OH S═O I I H I 3 CH(CH₃)CH(CH₃)OH S═O H CH₃ CH₃ CH₃ 3C(CH₃)₂CH₂OH S═O CH₃ CH₃ H CH₃ 3 CH₂C(CH₃)₂OH S═O H C₂H₅ C₂H₅ C₂H₅ 3CH₂CHF₂ S═O C₂H₅ C₂H₅ H C₂H₅ 3 CH₂CF₃ S═O H i- i- i- C₃H₇ C₃H₇ C₃H₇ 3CH₂CH(CH₃)₂ S═O i- i- H i- C₃H₇ C₃H₇ C₃H₇ 2 CH₂C(CH₃)₃ O═S═O H Cl Cl Cl2 H₂C—C≡N O═S═O Cl Cl H Cl 2 H₂C—C≡CH O═S═O H Br Br Br 2 CH(CH₃)₂ O═S═OBr Br H Br 2 CH₂CH₂OH O═S═O H I I I 2 CH₂CH(CH₃)OH O═S═O I I H I 3CH(CH₃)CH₂OH O═S═O H CH₃ CH₃ CH₃ 3 CH(CH₃)CH(CH₃)OH O═S═O CH₃ CH₃ H CH₃3 C(CH₃)₂CH₂OH O═S═O H C₂H₅ C₂H₅ C₂H₅ 3 CH₂C(CH₃)₂OH O═S═O C₂H₅ C₂H₅ HC₂H₅ 3 CH₂CHF₂ O═S═O H i- i- i- C₃H₇ C₃H₇ C₃H₇ 3 CH₂CF₃ O═S═O i- i- H i-C₃H₇ C₃H₇ C₃H₇ 2 CH₂CH(CH₃)₂ NH H Cl Cl Cl 2 CH₂C(CH₃)₃ NH Cl Cl H Cl 2H₂C—C≡N NH H Br Br Br 2 H₂C—C≡CH NH Br Br H Br 2 CH(CH₃)₂ NH H I I I 2CH₂CH₂OH NH I I H I 3 CH₂CH(CH₃)OH NH H CH₃ CH₃ CH₃ 3 CH(CH₃)CH₂OH NHCH₃ CH₃ H CH₃ 3 CH(CH₃)CH(CH₃)OH NH H C₂H₅ C₂H₅ C₂H₅ 3 C(CH₃)₂CH₂OH NHC₂H₅ C₂H₅ H C₂H₅ 3 CH₂C(CH₃)₂OH NH H i- i- i- C₃H₇ C₃H₇ C₃H₇ 3 CH₂CHF₂NH i- i- H i- C₃H₇ C₃H₇ C₃H₇ 2 CH₂CF₃ C═O H Cl Cl Cl 2 CH₂CH(CH₃)₂ C═OCl Cl H Cl 2 CH₂C(CH₃)₃ C═O H Br Br Br 2 H₂C—C≡N C═O Br Br H Br 2H₂C—C≡CH C═O H I I I 3 CH(CH₃)₂ C═O I I H I 3 CH₂CH₂OH C═O H CH₃ CH₃ CH₃3 CH₂CH(CH₃)OH C═O CH₃ CH₃ H CH₃ 3 CH(CH₃)CH₂OH C═O H C₂H₅ C₂H₅ C₂H₅ 3CH(CH₃)CH(CH₃)OH C═O C₂H₅ C₂H₅ H C₂H₅ 3 C(CH₃)₂CH₂OH C═O H i- i- i- C₃H₇C₃H₇ C₃H₇ 3 CH₂C(CH₃)₂OH C═O i- i- H i- C₃H₇ C₃H₇ C₃H₇ 2 CH₂CHF₂ C═S HCl Cl Cl 2 CH₂CF₃ C═S Cl Cl H Cl 2 CH₂CH(CH₃)₂ C═S H Br Br Br 2CH₂C(CH₃)₃ C═S Br Br H Br 2 H₂C—C≡N C═S H I I I 2 H₂C—C≡CH C═S I I H I 3CH(CH₃)₂ C═S H CH₃ CH₃ CH₃ 3 CH₂CH₂OH C═S CH₃ CH₃ H CH₃ 3 CH₂CH(CH₃)OHC═S H C₂H₅ C₂H₅ C₂H₅ 3 CH(CH₃)CH₂OH C═S C₂H₅ C₂H₅ H C₂H₅ 3CH(CH₃)CH(CH₃)OH C═S H i- i- i- C₃H₇ C₃H₇ C₃H₇ 3 C(CH₃)₂CH₂OH C═S i- i-H i- C₃H₇ C₃H₇ C₃H₇ 2 CH₂C(CH₃)₂OH CH₂ H Cl Cl Cl 2 CH₂CHF₂ CH₂ Cl Cl HCl 2 CH₂CF₃ CH₂ H Br Br Br 2 CH₂CH(CH₃)₂ CH₂ Br Br H Br 2 CH₂C(CH₃)₃ CH₂H I I I 2 H₂C—C≡N CH₂ I I H I 3 H₂C—C≡CH CH₂ H CH₃ CH₃ CH₃ 3 CH(CH₃)₂CH₂ CH₃ CH₃ H CH₃ 3 CH₂CH₂OH CH₂ H C₂H₅ C₂H₅ C₂H₅ 3 CH₂CH(CH₃)OH CH₂C₂H₅ C₂H₅ H C₂H₅ 3 CH(CH₃)CH₂OH CH₂ H i- i- i- C₃H₇ C₃H₇ C₃H₇ 3CH(CH₃)CH(CH₃)OH CH₂ i- i- H i- C₃H₇ C₃H₇ C₃H₇ 2 C(CH₃)₂CH₂OH CH—OH H ClCl Cl 2 CH₂C(CH₃)₂OH CH—OH Cl Cl H Cl 2 CH₂CHF₂ CH—OH H Br Br Br 2CH₂CF₃ CH—OH Br Br H Br 2 CH₂CH(CH₃)₂ CH—OH H I I I 2 CH₂C(CH₃)₃ CH—OH II H I 3 H₂C—C≡N CH—OH H CH₃ CH₃ CH₃ 3 H₂C—C≡CH CH—OH CH₃ CH₃ H CH₃ 3CH(CH₃)₂ CH—OH H C₂H₅ C₂H₅ C₂H₅ 3 CH₂CH₂OH CH—OH C₂H₅ C₂H₅ H C₂H₅ 3CH₂CH(CH₃)OH CH—OH H i- i- i- C₃H₇ C₃H₇ C₃H₇ 3 CH(CH₃)CH₂OH CH—OH i- i-H i- C₃H₇ C₃H₇ C₃H₇ 2 CH(CH₃)CH(CH₃)OH CH—F H Cl Cl Cl 2 C(CH₃)₂CH₂OHCH—F Cl Cl H Cl 2 CH₂C(CH₃)₂OH CH—F H Br Br Br 2 CH₂CHF₂ CH—F Br Br H Br2 CH₂CF₃ CH—F H I I I 2 CH₂CH(CH₃)₂ CH—F I I H I 3 CH₂C(CH₃)₃ CH—F H CH₃CH₃ CH₃ 3 H₂C—C≡N CH—F CH₃ CH₃ H CH₃ 3 H₂C—C≡CH CH—F H C₂H₅ C₂H₅ C₂H₅ 3CH(CH₃)₂ CH—F C₂H₅ C₂H₅ H C₂H₅ 3 CH₂CH₂OH CH—F H i- i- i- C₃H₇ C₃H₇ C₃H₇3 CH₂CH(CH₃)OH CH—F i- i- H i- C₃H₇ C₃H₇ C₃H₇ 2 CH(CH₃)CH₂OH S I H Cl H2 CH(CH₃)CH(CH₃)OH S═O I H H Cl 2 C(CH₃)₂CH₂OH O═S═O Br H Cl H 2CH₂C(CH₃)₂OH CH₂ Br H H Cl 2 CH₂CHF₂ C═O Br H I H 3 CH₂CF₃ C═S Br H H I3 CH₂CH(CH₃)₂ CH—OH I H Br H 3 CH₂C(CH₃)₃ CH—F I H H Br 3 H₂C—C≡N O I ClH Cl 3 H₂C—C≡CH NH Br Cl H Cl

TABLE 2B

and pharmaceutically acceptable salts thereof, where: m R³, R⁴ Y X² X³X⁴ X⁵ 2 aziridine S H Cl H Cl 2 azetidine S Cl H Cl H 2 pyrrolidine S ClH H Cl 2 piperazine S H Br H Br 2 morpholine S Br H Br H 2 piperidine SBr H H Br 2 N-methylpiperazine S H I H I 2 4-hydroxypiperidine S I H I H2 3-hydroxypiperidine S I H H I 2 4-aminopiperidine S H CH₃ H CH₃ 23-aminopiperidine S CH₃ H CH₃ H 2 3-hydroxypyrrolidne S CH₃ H H CH₃ 33-aminopyrrolidne S H C₂H₅ H C₂H₅ 3 aziridine S C₂H₅ H C₂H₅ H 3azetidine S C₂H₅ H H C₂H₅ 3 pyrrolidine S H i-C₃H₇ H i-C₃H₇ 3 piperazineS i-C₃H₇ H i-C₃H₇ H 3 morpholine S i-C₃H₇ H H i-C₃H₇ 3 piperidine O H ClH Cl 3 N-methylpiperazine O Cl H Cl H 3 4-hydroxypiperidine O Cl H H Cl3 3-hydroxypiperidine O H Br H Br 3 4-aminopiperidine O Br H Br H 33-aminopiperidine O Br H H Br 2 3-hydroxypyrrolidne O H I H I 23-aminopyrrolidne O I H I H 2 aziridine O I H H I 2 azetidine O H CH₃ HCH₃ 2 pyrrolidine O CH₃ H CH₃ H 2 piperazine O CH₃ H H CH₃ 2 morpholineO H C₂H₅ H C₂H₅ 2 piperidine O C₂H₅ H C₂H₅ H 2 N-methylpiperazine O C₂H₅H H C₂H₅ 2 4-hydroxypiperidine O H i-C₃H₇ H i-C₃H₇ 2 3-hydroxypiperidineO i-C₃H₇ H i-C₃H₇ H 2 4-aminopiperidine O i-C₃H₇ H H i-C₃H₇ 33-aminopiperidine S═O H Cl H Cl 3 3-hydroxypyrrolidne S═O Cl H Cl H 33-aminopyrrolidne S═O Cl H H Cl 3 aziridine S═O H Br H Br 3 azetidineS═O Br H Br H 3 pyrrolidine S═O Br H H Br 3 piperazine S═O H I H I 3morpholine S═O I H I H 3 piperidine S═O I H H I 3 N-methylpiperazine S═OH CH₃ H CH₃ 3 4-hydroxypiperidine S═O CH₃ H CH₃ H 3 3-hydroxypiperidineS═O CH₃ H H CH₃ 2 4-aminopiperidine S═O H C₂H₅ H C₂H₅ 23-aminopiperidine S═O C₂H₅ H C₂H₅ H 2 3-hydroxypyrrolidne S═O C₂H₅ H HC₂H₅ 2 3-aminopyrrolidne S═O H i-C₃H₇ H i-C₃H₇ 2 aziridine S═O i-C₃H₇ Hi-C₃H₇ H 2 azetidine S═O i-C₃H₇ H H i-C₃H₇ 2 pyrrolidine O═S═O H Cl H Cl2 piperazine O═S═O Cl H Cl H 2 morpholine O═S═O Cl H H Cl 2 piperidineO═S═O H Br H Br 2 N-methylpiperazine O═S═O Br H Br H 24-hydroxypiperidine O═S═O Br H H Br 3 3-hydroxypiperidine O═S═O H I H I3 4-aminopiperidine O═S═O I H I H 3 3-aminopiperidine O═S═O I H H I 33-hydroxypyrrolidne O═S═O H CH₃ H CH₃ 3 3-aminopyrrolidne O═S═O CH₃ HCH₃ H 3 aziridine O═S═O CH₃ H H CH₃ 3 azetidine O═S═O H C₂H₅ H C₂H₅ 3pyrrolidine O═S═O C₂H₅ H C₂H₅ H 3 piperazine O═S═O C₂H₅ H H C₂H₅ 3morpholine O═S═O H i-C₃H₇ H i-C₃H₇ 3 piperidine O═S═O i-C₃H₇ H i-C₃H₇ H3 N-methylpiperazine O═S═O i-C₃H₇ H H i-C₃H₇ 2 4-hydroxypiperidine NH HCl H Cl 2 3-hydroxypiperidine NH Cl H Cl H 2 4-aminopiperidine NH Cl H HCl 2 3-aminopiperidine NH H Br H Br 2 3-hydroxypyrrolidne NH Br H Br H 23-aminopyrrolidne NH Br H H Br 2 aziridine NH H I H I 2 azetidine NH I HI H 2 pyrrolidine NH I H H I 2 piperazine NH H CH₃ H CH₃ 2 morpholine NHCH₃ H CH₃ H 2 piperidine NH CH₃ H H CH₃ 3 N-methylpiperazine NH H C₂H₅ HC₂H₅ 3 4-hydroxypiperidine NH C₂H₅ H C₂H₅ H 3 3-hydroxypiperidine NHC₂H₅ H H C₂H₅ 3 4-aminopiperidine NH H i-C₃H₇ H i-C₃H₇ 33-aminopiperidine NH i-C₃H₇ H i-C₃H₇ H 3 3-hydroxypyrrolidne NH i-C₃H₇ HH i-C₃H₇ 3 3-aminopyrrolidne C═O H Cl H Cl 3 aziridine C═O Cl H Cl H 3azetidine C═O Cl H H Cl 3 pyrrolidine C═O H Br H Br 3 piperazine C═O BrH Br H 3 morpholine C═O Br H H Br 3 piperidine C═O H I H I 2N-methylpiperazine C═O I H I H 2 4-hydroxypiperidine C═O I H H I 23-hydroxypiperidine C═O H CH₃ H CH₃ 2 4-aminopiperidine C═O CH₃ H CH₃ H2 3-aminopiperidine C═O CH₃ H H CH₃ 2 3-hydroxypyrrolidne C═O H C₂H₅ HC₂H₅ 2 3-aminopyrrolidne C═O C₂H₅ H C₂H₅ H 2 aziridine C═O C₂H₅ H H C₂H₅2 azetidine C═O H i-C₃H₇ H i-C₃H₇ 2 pyrrolidine C═O i-C₃H₇ H i-C₃H₇ H 2piperazine C═O i-C₃H₇ H H i-C₃H₇ 3 morpholine C═S H Cl H Cl 3 piperidineC═S Cl H Cl H 3 N-methylpiperazine C═S Cl H H Cl 3 4-hydroxypiperidineC═S H Br H Br 3 3-hydroxypiperidine C═S Br H Br H 3 4-aminopiperidineC═S Br H H Br 3 3-aminopiperidine C═S H I H I 3 3-hydroxypyrrolidne C═SI H I H 3 3-aminopyrrolidne C═S I H H I 3 aziridine C═S H CH₃ H CH₃ 2azetidine C═S CH₃ H CH₃ H 2 pyrrolidine C═S CH₃ H H CH₃ 2 piperazine C═SH C₂H₅ H C₂H₅ 2 morpholine C═S C₂H₅ H C₂H₅ H 2 piperidine C═S C₂H₅ H HC₂H₅ 2 N-methylpiperazine C═S H i-C₃H₇ H i-C₃H₇ 2 4-hydroxypiperidineC═S i-C₃H₇ H i-C₃H₇ H 2 3-hydroxypiperidine C═S i-C₃H₇ H H i-C₃H₇ 34-aminopiperidine CH₂ H Cl H Cl 3 3-aminopiperidine CH₂ Cl H Cl H 33-hydroxypyrrolidne CH₂ Cl H H Cl 3 3-aminopyrrolidne CH₂ H Br H Br 3aziridine CH₂ Br H Br H 3 azetidine CH₂ Br H H Br 2 pyrrolidine CH₂ H IH I 2 piperazine CH₂ I H I H 2 morpholine CH₂ I H H I 2 piperidine CH₂ HCH₃ H CH₃ 2 N-methylpiperazine CH₂ CH₃ H CH₃ H 2 4-hydroxypiperidine CH₂CH₃ H H CH₃ 3 3-hydroxypiperidine CH₂ H C₂H₅ H C₂H₅ 3 4-aminopiperidineCH₂ C₂H₅ H C₂H₅ H 3 3-aminopiperidine CH₂ C₂H₅ H H C₂H₅ 33-hydroxypyrrolidne CH₂ H i-C₃H₇ H i-C₃H₇ 3 3-aminopyrrolidne CH₂ i-C₃H₇H i-C₃H₇ H 3 aziridine CH₂ i-C₃H₇ H H i-C₃H₇ 2 azetidine CH—OH H Cl H Cl2 pyrrolidine CH—OH Cl H Cl H 2 piperazine CH—OH Cl H H Cl 2 morpholineCH—OH H Br H Br 2 piperidine CH—OH Br H Br H 2 N-methylpiperazine CH—OHBr H H Br 3 4-hydroxypiperidine CH—OH H I H I 3 3-hydroxypiperidineCH—OH I H I H 3 4-aminopiperidine CH—OH I H H I 3 3-aminopiperidineCH—OH H CH₃ H CH₃ 3 3-hydroxypyrrolidne CH—OH CH₃ H CH₃ H 33-aminopyrrolidne CH—OH CH₃ H H CH₃ 2 aziridine CH—OH H C₂H₅ H C₂H₅ 2azetidine CH—OH C₂H₅ H C₂H₅ H 2 pyrrolidine CH—OH C₂H₅ H H C₂H₅ 2piperazine CH—OH H i-C₃H₇ H i-C₃H₇ 2 morpholine CH—OH i-C₃H₇ H i-C₃H₇ H2 piperidine CH—OH i-C₃H₇ H H i-C₃H₇ 3 N-methylpiperazine CH—F H Cl H Cl3 4-hydroxypiperidine CH—F Cl H Cl H 3 3-hydroxypiperidine CH—F Cl H HCl 3 4-aminopiperidine CH—F H Br H Br 3 3-aminopiperidine CH—F Br H Br H3 3-hydroxypyrrolidne CH—F Br H H Br 2 3-aminopyrrolidne CH—F H I H I 2aziridine CH—F I H I H 2 azetidine CH—F I H H I 2 pyrrolidine CH—F H CH₃H CH₃ 2 piperazine CH—F CH₃ H CH₃ H 2 morpholine CH—F CH₃ H H CH₃ 3piperidine CH—F H C₂H₅ H C₂H₅ 3 N-methylpiperazine CH—F C₂H₅ H C₂H₅ H 34-hydroxypiperidine CH—F C₂H₅ H H C₂H₅ 3 3-hydroxypiperidine CH—F Hi-C₃H₇ H i-C₃H₇ 3 4-aminopiperidine CH—F i-C₃H₇ H i-C₃H₇ H 33-aminopiperidine CH—F i-C₃H₇ H H i-C₃H₇ 2 3-hydroxypyrrolidne S H Cl ClCl 2 3-aminopyrrolidne S Cl Cl H Cl 2 aziridine S H Br Br Br 2 azetidineS Br Br H Br 2 pyrrolidine S H I I I 2 piperazine S I I H I 3 morpholineS H CH₃ CH₃ CH₃ 3 piperidine S CH₃ CH₃ H CH₃ 3 N-methylpiperazine S HC₂H₅ C₂H₅ C₂H₅ 3 4-hydroxypiperidine S C₂H₅ C₂H₅ H C₂H₅ 33-hydroxypiperidine S H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 4-aminopiperidine Si-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 3-aminopiperidine O H Cl Cl Cl 23-hydroxypyrrolidne O Cl Cl H Cl 2 3-aminopyrrolidne O H Br Br Br 2aziridine O Br Br H Br 2 azetidine O H I I I 2 pyrrolidine O I I H I 3piperazine O H CH₃ CH₃ CH₃ 3 morpholine O CH₃ CH₃ H CH₃ 3 piperidine O HC₂H₅ C₂H₅ C₂H₅ 3 N-methylpiperazine O C₂H₅ C₂H₅ H C₂H₅ 34-hydroxypiperidine O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 3-hydroxypiperidine Oi-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 4-aminopiperidine S═O H Cl Cl Cl 23-aminopiperidine S═O Cl Cl H Cl 2 3-hydroxypyrrolidne S═O H Br Br Br 23-aminopyrrolidne S═O Br Br H Br 2 aziridine S═O H I I I 2 aziridine S═OI I H I 3 azetidine S═O H CH₃ CH₃ CH₃ 3 pyrrolidine S═O CH₃ CH₃ H CH₃ 3piperazine S═O H C₂H₅ C₂H₅ C₂H₅ 3 morpholine S═O C₂H₅ C₂H₅ H C₂H₅ 3piperidine S═O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 N-methylpiperazine S═O i-C₃H₇i-C₃H₇ H i-C₃H₇ 2 4-hydroxypiperidine O═S═O H Cl Cl Cl 23-hydroxypiperidine O═S═O Cl Cl H Cl 2 4-aminopiperidine O═S═O H Br BrBr 2 3-aminopiperidine O═S═O Br Br H Br 2 3-hydroxypyrrolidne O═S═O H II I 2 3-aminopyrrolidne O═S═O I I H I 3 aziridine O═S═O H CH₃ CH₃ CH₃ 3azetidine O═S═O CH₃ CH₃ H CH₃ 3 pyrrolidine O═S═O H C₂H₅ C₂H₅ C₂H₅ 3piperazine O═S═O C₂H₅ C₂H₅ H C₂H₅ 3 morpholine O═S═O H i-C₃H₇ i-C₃H₇i-C₃H₇ 3 piperidine O=S=O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 N-methylpiperazine NHH Cl Cl Cl 2 4-hydroxypiperidine NH Cl Cl H Cl 2 3-hydroxypiperidine NHH Br Br Br 2 4-aminopiperidine NH Br Br H Br 2 3-aminopiperidine NH H II I 2 3-hydroxypyrrolidne NH I I H I 3 3-aminopyrrolidne NH H CH₃ CH₃CH₃ 3 aziridine NH CH₃ CH₃ H CH₃ 3 azetidine NH H C₂H₅ C₂H₅ C₂H₅ 3pyrrolidine NH C₂H₅ C₂H₅ H C₂H₅ 3 piperazine NH H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3morpholine NH i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 piperidine C═O H Cl Cl Cl 2N-methylpiperazine C═O Cl Cl H Cl 2 4-hydroxypiperidine C═O H Br Br Br 23-hydroxypiperidine C═O Br Br H Br 2 4-aminopiperidine C═O H I I I 33-aminopiperidine C═O I I H I 3 3-hydroxypyrrolidne C═O H CH₃ CH₃ CH₃ 33-aminopyrrolidne C═O CH₃ CH₃ H CH₃ 3 aziridine C═O H C₂H₅ C₂H₅ C₂H₅ 3azetidine C═O C₂H₅ C₂H₅ H C₂H₅ 3 pyrrolidine C═O H i-C₃H₇ i-C₃H₇ i-C₃H₇3 piperazine C═O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 morpholine C═S H Cl Cl Cl 2piperidine C═S Cl Cl H Cl 2 N-methylpiperazine C═S H Br Br Br 24-hydroxypiperidine C═S Br Br H Br 2 3-hydroxypiperidine C═S H I I I 24-aminopiperidine C═S I I H I 3 3-aminopiperidine C═S H CH₃ CH₃ CH₃ 33-hydroxypyrrolidne C═S CH₃ CH₃ H CH₃ 3 3-aminopyrrolidne C═S H C₂H₅C₂H₅ C₂H₅ 3 aziridine C═S C₂H₅ C₂H₅ H C₂H₅ 3 azetidine C═S H i-C₃H₇i-C₃H₇ i-C₃H₇ 3 pyrrolidine C═S i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 piperazine CH₂H Cl Cl Cl 2 morpholine CH₂ Cl Cl H Cl 2 piperidine CH₂ H Br Br Br 2N-methylpiperazine CH₂ Br Br H Br 2 4-hydroxypiperidine CH₂ H I I I 23-hydroxypiperidine CH₂ I I H I 3 4-aminopiperidine CH₂ H CH₃ CH₃ CH₃ 33-aminopiperidine CH₂ CH₃ CH₃ H CH₃ 3 3-hydroxypyrrolidne CH₂ H C₂H₅C₂H₅ C₂H₅ 3 3-aminopyrrolidne CH₂ C₂H₅ C₂H₅ H C₂H₅ 3 aziridine CH₂ Hi-C₃H₇ i-C₃H₇ i-C₃H₇ 3 azetidine CH₂ i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2pyrrolidine CH—OH H Cl Cl Cl 2 piperazine CH—OH Cl Cl H Cl 2 morpholineCH—OH H Br Br Br 2 piperidine CH—OH Br Br H Br 2 N-methylpiperazineCH—OH H I I I 2 4-hydroxypiperidine CH—OH I I H I 3 3-hydroxypiperidineCH—OH H CH₃ CH₃ CH₃ 3 4-aminopiperidine CH—OH CH₃ CH₃ H CH₃ 33-aminopiperidine CH—OH H C₂H₅ C₂H₅ C₂H₅ 3 3-hydroxypyrrolidne CH—OHC₂H₅ C₂H₅ H C₂H₅ 3 3-aminopyrrolidne CH—OH H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3aziridine CH—OH i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 azetidine CH—F H Cl Cl Cl 2pyrrolidine CH—F Cl Cl H Cl 2 piperazine CH—F H Br Br Br 2 morpholineCH—F Br Br H Br 2 piperidine CH—F H I I I 2 N-methylpiperazine CH—F I IH I 3 4-hydroxypiperidine CH—F H CH₃ CH₃ CH₃ 3 3-hydroxypiperidine CH—FCH₃ CH₃ H CH₃ 3 4-aminopiperidine CH—F H C₂H₅ C₂H₅ C₂H₅ 33-aminopiperidine CH—F C₂H₅ C₂H₅ H C₂H₅ 3 3-hydroxypyrrolidne CH—F Hi-C₃H₇ i-C₃H₇ i-C₃H₇ 3 3-aminopyrrolidne CH—F i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2aziridine S I H Cl H 2 azetidine S—O I H H Cl 2 pyrrolidine O═S═O Br HCl H 2 piperazine CH₂ Br H H Cl 2 morpholine C═O Br H I H 3 piperidineC═S Br H H I 3 N-methylpiperazine CH—OH I H Br H 3 4-hydroxypiperidineCH—F I H H Br 3 3-hydroxypiperidine O I Cl H Cl 3 4-aminopiperidine NHBr Cl H Cl

TABLE 2C

and pharmaceutically acceptable salts thereof, where: m R⁴ R⁶ Y X² X³ X⁴X⁵ 1 CH(CH₃)₂ CH₃ S H Cl H Cl 1 CH₂CH₂OH CH₃ S Cl H Cl H 1 CH₂CH(CH₃)OHC₂H₅ S Cl H H Cl 1 CH(CH₃)CH₂OH C₂H₅ S H Br H Br 1 CH(CH₃)CH(CH₃)OHn-C₃H₇ S Br H Br H 1 C(CH₃)₂CH₂OH n-C₃H₇ S Br H H Br 1 CH₂C(CH₃)₂OHi-C₃H₇ S H I H I 1 CH₂CHF₂ i-C₃H₇ S I H I H 1 CH₂CF₃ c-C₃H₅ S I H H I 2CH₂CH(CH₃)₂ c-C₃H₅ S H CH₃ H CH₃ 2 CH₂C(CH₃)₃ sec-C₄H₉ S CH₃ H CH₃ H 2H₂C—C≡N sec-C₄H₉ S CH₃ H H CH₃ 2 H₂C—C≡CH i-C₄H₉ S H C₂H₅ H C₂H₅ 2CH(CH₃)₂ i-C₄H₉ S C₂H₅ H C₂H₅ H 2 CH₂CH₂OH n-C₄H₉ S C₂H₅ H H C₂H₅ 2CH₂CH(CH₃)OH n-C₄H₉ S H i-C₃H₇ H i-C₃H₇ 2 CH(CH₃)CH₂OH CH₃ S i-C₃H₇ Hi-C₃H₇ H 2 CH(CH₃)CH(CH₃)OH CH₃ S i-C₃H₇ H H i-C₃H₇ 1 C(CH₃)₂CH₂OH C₂H₅O H Cl H Cl 1 CH₂C(CH₃)₂OH C₂H₅ O Cl H Cl H 1 CH₂CHF₂ n-C₃H₇ O Cl H H Cl1 CH₂CF₃ n-C₃H₇ O H Br H Br 1 CH₂CH(CH₃)₂ i-C₃H₇ O Br H Br H 1CH₂C(CH₃)₃ i-C₃H₇ O Br H H Br 1 H₂C—C≡N c-C₃H₅ O H I H I 1 H₂C—C≡CHc-C₃H₅ O I H I H 1 CH(CH₃)₂ sec-C₄H₉ O I H H I 2 CH₂CH₂OH sec-C₄H₉ O HCH₃ H CH₃ 2 CH₂CH(CH₃)OH i-C₄H₉ O CH₃ H CH₃ H 2 CH(CH₃)CH₂OH i-C₄H₉ OCH₃ H H CH₃ 2 CH(CH₃)CH(CH₃)OH n-C₄H₉ O H C₂H₅ H C₂H₅ 2 C(CH₃)₂CH₂OHn-C₄H₉ O C₂H₅ H C₂H₅ H 2 CH₂C(CH₃)₂OH CH₃ O C₂H₅ H H C₂H₅ 2 CH₂CHF₂ CH₃O H i-C₃H₇ H i-C₃H₇ 2 CH₂CF₃ C₂H₅ O i-C₃H₇ H i-C₃H₇ H 2 CH₂CH(CH₃)₂ C₂H₅O i-C₃H₇ H H i-C₃H₇ 1 CH₂C(CH₃)₃ n-C₃H₇ S═O H Cl H Cl 1 H₂C—C≡N n-C₃H₇S═O Cl H Cl H 1 H₂C—C≡CH i-C₃H₇ S═O Cl H H Cl 1 CH(CH₃)₂ i-C₃H₇ S═O H BrH Br 1 CH₂CH₂OH c-C₃H₅ S═O Br H Br H 1 CH₂CH(CH₃)OH c-C₃H₅ S═O Br H H Br1 CH(CH₃)CH₂OH sec-C₄H₉ S═O H I H I 1 CH(CH₃)CH(CH₃)OH sec-C₄H₉ S═O I HI H 1 C(CH₃)₂CH₂OH i-C₄H₉ S═O I H H I 2 CH₂C(CH₃)₂OH i-C₄H₉ S═O H CH₃ HCH₃ 2 CH₂CHF₂ n-C₄H₉ S═O CH₃ H CH₃ H 2 CH₂CF₃ n-C₄H₉ S═O CH₃ H H CH₃ 2CH₂CH(CH₃)₂ CH₃ S═O H C₂H₅ H C₂H₅ 2 CH₂C(CH₃)₃ CH₃ S═O C₂H₅ H C₂H₅ H 2H₂C—C≡N C₂H₅ S═O C₂H₅ H H C₂H₅ 2 H₂C—C≡CH C₂H₅ S═O H i-C₃H₇ H i-C₃H₇ 2CH(CH₃)₂ n-C₃H₇ S═O i-C₃H₇ H i-C₃H₇ H 2 CH₂CH₂OH n-C₃H₇ S═O i-C₃H₇ H Hi-C₃H₇ 1 CH₂CH(CH₃)OH i-C₃H₇ O═S═O H Cl H Cl 1 CH(CH₃)CH₂OH i-C₃H₇ O═S═OCl H Cl H 1 CH(CH₃)CH(CH₃)OH c-C₃H₅ O═S═O Cl H H Cl 1 C(CH₃)₂CH₂OHc-C₃H₅ O═S═O H Br H Br 1 CH₂C(CH₃)₂OH sec-C₄H₉ O═S═O Br H Br H 1 CH₂CHF₂sec-C₄H₉ O═S═O Br H H Br 1 CH₂CF₃ i-C₄H₉ O═S═O H I H I 1 CH₂CH(CH₃)₂i-C₄H9 O═S═O I H I H 1 CH₂C(CH₃)₃ n-C₄H₉ O═S═O I H H I 2 H₂C—C≡N n-C₄H₉O═S═O H CH₃ H CH₃ 2 H₂C—C≡CH CH₃ O═S═O CH₃ H CH₃ H 2 CH(CH₃)₂ CH₃ O═S═OCH₃ H H CH₃ 2 CH₂CH₂OH C₂H₅ O═S═O H C₂H₅ H C₂H₅ 2 CH₂CH(CH₃)OH C₂H₅O═S═O C₂H₅ H C₂H₅ H 2 CH(CH₃)CH₂OH n-C₃H₇ O═S═O C₂H₅ H H C₂H₅ 2CH(CH₃)CH(CH₃)OH n-C₃H₇ O═S═O H i-C₃H₇ H i-C₃H₇ 2 C(CH₃)₂CH₂OH i-C₃H₇O═S═O i-C₃H₇ H i-C₃H₇ H 2 CH₂C(CH₃)₂OH i-C₃H₇ O═S═O i-C₃H₇ H H i-C₃H₇ 1CH₂CHF₂ c-C₃H₅ NH H Cl H Cl 1 CH₂CF₃ c-C₃H₅ NH Cl H Cl H 1 CH₂CH(CH₃)₂sec-C₄H₉ NH Cl H H Cl 1 CH₂C(CH₃)₃ sec-C₄H₉ NH H Br H Br 1 H₂C—C≡Ni-C₄H₉ NH Br H Br H 1 H₂C—C≡CH i-C₄H₉ NH Br H H Br 1 CH(CH₃)₂ n-C₄H₉ NHH I H I 1 CH₂CH₂OH n-C₄H₉ NH I H I H 1 CH₂CH(CH₃)OH CH₃ NH I H H I 2CH(CH₃)CH₂OH CH₃ NH H CH₃ H CH₃ 2 CH(CH₃)CH(CH₃)OH C₂H₅ NH CH₃ H CH₃ H 2C(CH₃)₂CH₂OH C₂H₅ NH CH₃ H H CH₃ 2 CH₂C(CH₃)₂OH n-C₃H₇ NH H C₂H₅ H C₂H₅2 CH₂CHF₂ n-C₃H₇ NH C₂H₅ H C₂H₅ H 2 CH₂CF₃ i-C₃H₇ NH C₂H₅ H H C₂H₅ 2CH₂CH(CH₃)₂ i-C₃H₇ NH H i-C₃H₇ H i-C₃H₇ 2 CH₂C(CH₃)₃ c-C₃H₅ NH i-C₃H₇ Hi-C₃H₇ H 2 H₂C—C≡N c-C₃H₅ NH i-C₃H₇ H H i-C₃H₇ 1 H₂C—C≡CH sec-C₄H₉ C═O HCl H Cl 1 CH(CH₃)₂ sec-C₄H₉ C═O Cl H Cl H 1 CH₂CH₂OH i-C₄H₉ C═O Cl H HCl 1 CH₂CH(CH₃)OH i-C₄H₉ C═O H Br H Br 1 CH(CH₃)CH₂OH n-C₄H₉ C═O Br H BrH 1 CH(CH₃)CH(CH₃)OH n-C₄H₉ C═O Br H H Br 1 C(CH₃)₂CH₂OH CH₃ C═O H I H I1 CH₂C(CH₃)₂OH CH₃ C═O I H I H 1 CH₂CHF₂ C₂H₅ C═O I H H I 2 CH₂CF₃ C₂H₅C═O H CH₃ H CH₃ 2 CH₂CH(CH₃)₂ n-C₃H₇ C═O CH₃ H CH₃ H 2 CH₂C(CH₃)₃ n-C₃H₇C═O CH₃ H H CH₃ 2 H₂C—C≡N i-C₃H₇ C═O H C₂H₅ H C₂H₅ 2 H₂C—C≡CH i-C₃H₇ C═OC₂H₅ H C₂H₅ H 2 CH(CH₃)₂ c-C₃H₅ C═O C₂H₅ H H C₂H₅ 2 CH₂CH₂OH c-C₃H₅ C═OH i-C═3H═7 H i-C₃H₇ 2 CH₂CH(CH₃)OH sec-C₄H₉ C═O i-C₃H₇ H i-C₃H₇ H 2CH(CH₃)CH₂OH sec-C₄H₉ C═O i-C₃H₇ H H i-C₃H₇ 1 CH(CH₃)CH(CH₃)OH i-C₄H₉C═S H Cl H Cl 1 C(CH₃)2CH₂OH i-C₄H₉ C═S Cl H Cl H 1 CH₂C(CH₃)₂OH n-C₄H₉C═S Cl H H Cl 1 CH₂CHF₂ n-C₄H₉ C═S H Br H Br 1 CH₂CF₃ CH₃ C═S Br H Br H1 CH₂CH(CH₃)₂ CH₃ C═S Br H H Br 1 CH₂C(CH₃)₃ C₂H₅ C═S H I H I 1 H₂C—C≡NC₂H₅ C═S I H I H 1 H₂C—C≡CH n-C₃H₇ C═S I H H I 2 CH(CH₃)₂ n-C₃H₇ C═S HCH₃ H CH₃ 2 CH₂CH₂OH i-C₃H₇ C═S CH H CH₃ H 2 CH₂CH(CH₃)OH i-C₃H₇ C═S CH₃H H CH₃ 2 CH(CH₃)CH₂OH c-C₃H₅ C═S H C₂H₅ H C₂H₅ 2 CH(CH₃)CH(CH₃)OHc-C₃H₅ C═S C₂H₅ H C₂H₅ H 2 C(CH₃)₂CH₂OH sec-C₄H₉ C═S C₂H₅ H H C₂H₅ 2CH₂C(CH₃)₂OH sec-C₄H₉ C═S H i-C₃H₇ H i-C₃H₇ 2 CH₂CHF₂ i-C₄H₉ C═S i-C₃H₇H i-C₃H₇ H 2 CH₂CF₃ i-C₄H₉ C═S i-C₃H₇ H H i-C₃H₇ 1 CH₂CH(CH₃)₂ n-C₄H₉CH₂ H Cl H Cl 1 CH₂C(CH₃)₃ n-C₄H₉ CH₂ Cl H Cl H 1 H₂C—C≡N CH₃ CH₂ Cl H HCl 1 H₂C—C≡CH CH₃ CH₂ H Br H Br 1 CH(CH₃)₂ C₂H₅ CH₂ Br H Br H 1 CH₂CH₂OHC₂H₅ CH₂ Br H H Br 1 CH₂CH(CH₃)OH n-C₃H₇ CH₂ H I H I 1 CH(CH₃)CH₂OHn-C₃H₇ CH₂ I H I H 1 CH(CH₃)CH(CH₃)OH i-C₃H₇ CH₂ I H H I 2 C(CH₃)₂CH₂OHi-C₃H₇ CH₂ H CH₃ H CH₃ 2 CH₂C(CH₃)₂OH c-C₃H₅ CH₂ CH₃ H CH₃ H 2 CH₂CHF₂c-C₃H₅ CH₂ CH₃ H H CH₃ 2 CH₂CF₃ sec-C₄H₉ CH₂ H C₂H₅ H C₂H₅ 2 CH₂CH(CH₃)₂sec-C₄H₉ CH₂ C₂H₅ H C₂H₅ H 2 CH₂C(CH₃)₃ i-C₄H₉ CH₂ C₂H₅ H H C₂H₅ 2H₂C—C≡N i-C₄H₉ CH₂ H i-C₃H₇ H i-C₃H₇ 2 H₂C—C≡CH n-C₄H₉ CH₂ i-C₃H₇ Hi-C₃H₇ H 2 CH(CH₃)₂ n-C₄H₉ CH₂ i-C₃H₇ H H i-C₃H₇ 1 CH₂CH₂OH CH₃ CH—OH HCl H Cl 1 CH₂CH(CH₃)OH CH₃ CH—OH Cl H Cl H 1 CH(CH₃)CH₂OH C₂H₅ CH—OH ClH H Cl 1 CH(CH₃)CH(CH₃)OH C₂H₅ CH—OH H Br H Br 1 C(CH₃)₂CH₂OH n-C₃H₇CH—OH Br H Br H 1 CH₂C(CH₃)₂OH n-C₃H₇ CH—OH Br H H Br 1 CH₂CHF₂ i-C₃H₇CH—OH H I H I 1 CH₂CF₃ i-C₃H₇ CH—OH I H I H 1 CH₂CH(CH₃)₂ c-C₃H₅ CH—OH IH H I 2 CH₂C(CH₃)₃ c-C₃H₅ CH—OH H CH₃ H CH₃ 2 H₂C—C≡N sec-C₄H₉ CH—OH CH₃H CH₃ H 2 H₂C—C≡CH sec-C₄H₉ CH—OH CH₃ H H CH₃ 2 CH(CH₃)₂ i-C₄H₉ CH—OH HC₂H₅ H C₂H₅ 2 CH₂CH₂OH i-C₄H₉ CH—OH C₂H₅ H C₂H₅ H 2 CH₂CH(CH₃)OH n-C₄H₉CH—OH C₂H₅ H H C₂H₅ 2 CH(CH₃)CH₂OH n-C₄H₉ CH—OH H i-C₃H₇ H i-C₃H₇ 2CH(CH₃)CH(CH₃)OH CH₃ CH—OH i-C₃H₇ H i-C₃H₇ H 2 C(CH₃)₂CH₂OH CH₃ CH—OHi-C₃H₇ H H i-C₃H₇ 1 CH₂C(CH₃)₂OH C₂H₅ CH—F H Cl H Cl 1 CH₂CHF₂ C₂H₅ CH—FCl H Cl H 1 CH₂CF₃ n-C₃H₇ CH—F Cl H H Cl 1 CH₂CH(CH₃)₂ n-C₃H₇ CH—F H BrH Br 1 CH₂C(CH₃)₃ i-C₃H₇ CH—F Br H Br H 1 H₂C—C≡N i-C₃H₇ CH—F Br H H Br1 H₂C—C≡CH c-C₃H₅ CH—F H I H I 1 CH(CH₃)₂ c-C₃H₅ CH—F I H I H 1 CH₂CH₂OHsec-C₄H₉ CH—F I H H I 2 CH₂CH(CH₃)OH sec-C₄H₉ CH—F H CH₃ H CH₃ 2CH(CH₃)CH₂OH i-C₄H₉ CH—F CH₃ H CH₃ H 2 CH(CH₃)CH(CH₃)OH i-C₄H₉ CH—F CH₃H H CH₃ 2 C(CH₃)₂CH₂OH n-C₄H₉ CH—F H C₂H₅ H C₂H₅ 2 CH₂C(CH₃)₂OH n-C₄H₉CH—F C₂H₅ H C₂H₅ H 2 CH₂CHF₂ CH₃ CH—F C₂H₅ H H C₂H₅ 2 CH₂CF₃ CH₃ CH—F Hi-C₃H₇ H i-C₃H₇ 2 CH₂CH(CH₃)₂ C₂H₅ CH—F i-C₃H₇ H i-C₃H₇ H 2 CH₂C(CH₃)₃C₂H₅ CH—F i-C₃H₇ H H i-C₃H₇ 1 H₂C—C≡N n-C₃H₇ S H Cl Cl Cl 1 H₂C—C≡CHn-C₃H₇ S Cl Cl H Cl 1 CH(CH₃)₂ i-C₃H₇ S H Br Br Br 1 CH₂CH₂OH i-C₃H₇ SBr Br H Br 1 CH₂CH(CH₃)OH c-C₃H₅ S H I I I 1 CH(CH₃)CH₂OH c-C₃H₅ S I I HI 1 CH(CH₃)CH(CH₃)OH sec-C₄H₉ S H CH₃ CH₃ CH₃ 1 C(CH₃)₂CH₂OH sec-C₄H₉ SCH₃ CH₃ H CH₃ 1 CH₂C(CH₃)₂OH i-C₄H₉ S H C₂H₅ C₂H₅ C₂H₅ 2 CH₂CHF₂ i-C₄H₉S C₂H₅ C₂H₅ H C₂H₅ 2 CH₂CF₃ n-C₄H₉ S H i-C₃H₇ i-C₃H₇ i-C₃H₇ 2CH₂CH(CH₃)₂ n-C₄H₉ S i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 CH₂C(CH₃)₃ CH₃ O H Cl ClCl 2 H₂C—C≡N CH₃ O Cl Cl H Cl 2 H₂C—C≡CH C₂H₅ O H Br Br Br 2 CH(CH₃)₂C₂H₅ O Br Br H Br 2 CH₂CH₂OH n-C₃H₇ O H I I I 2 CH₂CH(CH₃)OH n-C₃H₇ O II H I 1 CH(CH₃)CH₂OH i-C₃H₇ O H CH₃ CH₃ CH₃ 1 CH(CH₃)CH(CH₃)OH i-C₃H₇ OCH₃ CH₃ H CH₃ 1 C(CH₃)₂CH₂OH c-C₃H₅ O H C₂H₅ C₂H₅ C₂H₅ 1 CH₂C(CH₃)₂OHc-C₃H₅ O C₂H₅ C₂H₅ H C₂H₅ 1 CH₂CHF₂ sec-C₄H₉ O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 1CH₂CF₃ sec-C₄H₉ O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 1 CH₂CH(CH₃)₂ i-C₄H₉ S═O H ClCl Cl 1 CH₂C(CH₃)₃ i-C₄H₉ S═O Cl Cl H Cl 1 H₂C—C≡N n-C₄H₉ S═O H Br Br Br2 H₂C—C≡CH n-C₄H₉ S═O Br Br H Br 2 CH(CH₃)₂ CH₃ S═O H I I I 2 CH₂CH₂OHCH₃ S═O I I H I 2 CH₂CH(CH₃)OH C₂H₅ S═O H CH₃ CH₃ CH₃ 2 CH(CH₃)CH₂OHC₂H₅ S═O CH₃ CH₃ H CH₃ 2 CH(CH₃)CH(CH₃)OH n-C₃H₇ S═O H C₂H₅ C₂H₅ C₂H₅ 2C(CH₃)₂CH₂OH n-C₃H₇ S═O C₂H₅ C₂H₅ H C₂H₅ 2 CH₂C(CH₃)₂OH i-C₃H₇ S═O Hi-C₃H₇ i-C₃H₇ i-C₃H₇ 2 CH₂CHF₂ i-C₃H₇ S═O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 1CH₂CF₃ c-C₃H₅ O═S═O H Cl Cl Cl 1 CH₂CH(CH₃)₂ c-C₃H₅ O═S═O Cl Cl H Cl 1CH₂C(CH₃)₃ sec-C₄H₉ O═S═O H Br Br Br 1 H₂C—C≡N sec-C₄H₉ O═S═O Br Br H Br1 H₂C—C≡CH i-C₄H₉ O═S═O H I I I 1 CH(CH₃)₂ i-C₄H₉ O═S═O I I H I 1CH₂CH₂OH n-C₄H₉ O═S═O H CH₃ CH₃ CH₃ 1 CH₂CH(CH₃)OH n-C₄H₉ O═S═O CH₃ CH₃H CH₃ 1 CH(CH₃)CH₂OH CH₃ O═S═O H C₂H₅ C₂H₅ C₂H₅ 2 CH(CH₃)CH(CH₃)OH CH₃O═S═O C₂H₅ C₂H₅ H C₂H₅ 2 C(CH₃)₂CH₂OH C₂H₅ O═S═O H i-C₃H₇ i-C₃H₇ i-C₃H₇2 CH₂C(CH₃)₂OH C₂H₅ O═S═O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 CH₂CHF₂ n-C₃H₇ NH HCl Cl Cl 2 CH₂CF₃ n-C₃H₇ NH Cl Cl H Cl 2 CH₂CH(CH₃)₂ i-C₃H₇ NH H Br BrBr 2 CH₂C(CH₃)₃ i-C₃H₇ NH Br Br H Br 2 H₂C—C≡N c-C₃H₅ NH H I I I 2H₂C—C≡CH c-C₃H₅ NH I I H I 1 CH(CH₃)₂ sec-C₄H₉ NH H CH₃ CH₃ CH₃ 1CH₂CH₂OH sec-C₄H₉ NH CH₃ CH₃ H CH₃ 1 CH₂CH(CH₃)OH i-C₄H₉ NH H C₂H₅ C₂H₅C₂H₅ 1 CH(CH₃)CH₂OH i-C₄H₉ NH C₂H₅ C₂H₅ H C₂H₅ 1 CH(CH₃)CH(CH₃)OH n-C₄H₉NH H i-C₃H₇ i-C₃H₇ i-C₃H₇ 1 C(CH₃)₂CH₂OH n-C₄H₉ NH i-C₃H₇ i-C₃H₇ Hi-C₃H₇ 1 CH₂C(CH₃)₂OH CH₃ C═O H Cl Cl Cl 1 CH₂CHF₂ CH₃ C═O Cl Cl H Cl 1CH₂CF₃ C₂H₅ C═O H Br Br Br 2 CH₂CH(CH₃)₂ C₂H₅ C═O Br Br H Br 2CH₂C(CH₃)₃ n-C₃H₇ C═O H I I I 2 H₂C—C≡N n-C₃H₇ C═O I I H I 2 H₂C—C≡CHi-C₃H₇ C═O H CH₃ CH₃ CH₃ 2 CH(CH₃)₂ i-C₃H₇ C═O CH₃ CH₃ H CH₃ 2 CH₂CH₂OHc-C₃H₅ C═O H C₂H₅ C₂H₅ C₂H₅ 2 CH₂CH(CH₃)OH c-C₃H₅ C═O C₂H₅ C₂H₅ H C₂H₅ 2CH(CH₃)CH₂OH sec-C₄H₉ C═O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 2 CH(CH₃)CH(CH₃)OHsec-C₄H₉ C═O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 1 C(CH₃)₂CH₂OH i-C₄H₉ C═S H Cl Cl Cl1 CH₂C(CH₃)₂OH i-C₄H₉ C═S Cl Cl H Cl 1 CH₂CHF₂ n-C₄H₉ C═S H Br Br Br 1CH₂CF₃ n-C₄H₉ C═S Br Br H Br 1 CH₂CH(CH₃)₂ CH₃ C═S H I I I 1 CH₂C(CH₃)₃CH₃ C═S I I H I 1 H₂C—C≡N C₂H₅ C═S H CH₃ CH₃ CH₃ 1 H₂C—C≡CH C₂H₅ C═S CHCH₃ H CH₃ 1 CH(CH₃)₂ n-C₃H₇ C═S H C₂H₅ C₂H₅ C₂H₅ 2 CH₂CH₂OH n-C₃H₇ C═SC₂H₅ C₂H₅ H C₂H₅ 2 CH₂CH(CH₃)OH i-C₃H₇ C═S H i-C₃H₇ i-C₃H₇ i-C₃H₇ 2CH(CH₃)CH₂OH i-C₃H₇ C═S i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 CH(CH₃)CH(CH₃)OH c-C₃H₅CH₂ H Cl Cl Cl 2 C(CH₃)₂CH₂OH c-C₃H₅ CH₂ Cl Cl H Cl 2 CH₂C(CH₃)₂OHsec-C₄H₉ CH₂ H Br Br Br 2 CH₂CHF₂ sec-C₄H₉ CH₂ Br Br H Br 2 CH₂CF₃i-C₄H₉ CH₂ H I I I 2 CH₂CH(CH₃)₂ i-C₄H₉ CH₂ I I H I 1 CH₂C(CH₃)₃ n-C₄H₉CH₂ H CH₃ CH₃ CH₃ 1 H₂C—C≡N n-C₄H₉ CH₂ CH₃ CH₃ H CH₃ 1 H₂C—C≡CH CH₃ CH₂H C₂H₅ C₂H₅ C₂H₅ 1 CH(CH₃)₂ CH₃ CH₂ C₂H₅ C₂H₅ H C₂H₅ 1 CH₂CH₂OH C₂H₅ CH₂H i-C₃H₇ i-C₃H₇ i-C₃H₇ 1 CH₂CH(CH₃)OH C₂H₅ CH₂ i-C₃H₇ i-C₃H₇ H i-C₃H₇ 1CH(CH₃)CH₂OH n-C₃H₇ CH—OH H Cl Cl Cl 1 CH(CH₃)CH(CH₃)OH n-C₃H₇ CH—OH ClCl H Cl 1 C(CH₃)₂CH₂OH i-C₃H₇ CH—OH H Br Br Br 2 CH₂C(CH₃)₂OH i-C₃H₇CH—OH Br Br H Br 2 CH₂CHF₂ c-C₃H₅ CH—OH H I I I 2 CH₂CF₃ c-C₃H₅ CH—OH II H I 2 CH₂CH(CH₃)₂ sec-C₄H₉ CH—OH H CH₃ CH₃ CH₃ 2 CH₂C(CH₃)₃ sec-C₄H₉CH—OH CH₃ CH₃ H CH₃ 2 H₂C—C≡N i-C₄H₉ CH—OH H C₂H₅ C₂H₅ C₂H₅ 2 H₂C—C≡CHi-C₄H₉ CH—OH C₂H₅ C₂H₅ H C₂H₅ 2 CH(CH₃)₂ n-C₄H₉ CH—OH H i-C₃H₇ i-C₃H₇i-C₃H₇ 2 CH₂CH₂OH n-C₄H₉ CH—OH i-C₃H₇ i-C₃H₇ H i-C₃H₇ 1 CH₂CH(CH₃)OH CH₃CH—F H Cl Cl Cl 1 CH(CH₃)CH₂OH CH₃ CH—F Cl Cl H Cl 1 CH(CH₃)CH(CH₃)OHC₂H₅ CH—F H Br Br Br 1 C(CH₃)₂CH₂OH C₂H₅ CH—F Br Br H Br 1 CH₂C(CH₃)₂OHn-C₃H₇ CH—F H I I I 1 CH₂CHF₂ n-C₃H₇ CH—F I I H I 1 CH₂CF₃ i-C₃H₇ CH—F HCH₃ CH₃ CH₃ 1 CH₂CH(CH₃)₂ i-C₃H₇ CH—F CH₃ CH₃ H CH₃ 1 CH₂C(CH₃)₃ c-C₃H₅CH—F H C₂H₅ C₂H₅ C₂H₅ 2 H₂C—C≡N c-C₃H₅ CH—F C₂H₅ C₂H₅ H C₂H₅ 2 H₂C—C≡CHsec-C₄H₉ CH—F H i-C₃H₇ i-C₃H₇ i-C₃H₇ 2 CH(CH₃)₂ sec-C₄H₉ CH—F i-C₃H₇i-C₃H₇ H i-C₃H₇ 2 CH₂CH₂OH i-C₄H₉ S I H Cl H 2 CH₂CH(CH₃)OH i-C₄H₉ S═O IH H Cl 2 CH(CH₃)CH₂OH n-C₄H₉ O═S═O Br H Cl H 2 CH(CH₃)CH(CH₃)OH n-C₄H₉CH₂ Br H H Cl 2 C(CH₃)₂CH₂OH CH₃ C═O Br H I H 2 CH₂C(CH₃)₂OH CH₃ C═S BrH H I 1 CH₂CHF₂ C₂H₅ CH—OH I H Br H 1 CH₂CF₃ C₂H₅ CH—F I H H Br 1CH₂CH(CH₃)2 n-C₃H₇ O I Cl H Cl 1 CH₂C(CH₃)₃ n-C₃H₇ NH Br Cl H Cl

TABLE 2D

m R⁴ Y X² X³ X⁴ X⁵ 2 CH₂CH(CH₃)OH S Furan-2-yl Cl H Cl 2 CH(CH₃)CH₂OH SFuran-3-yl H Cl H 2 CH(CH₃)CH(CH₃)OH S Furan-4-yl H H Cl 2 C(CH₃)₂CH₂OHS Furan-5-yl Br H Br 2 CH₂C(CH₃)₂OH S Pyrrol-2-yl H Br H 2 CH₂CHF₂ SPyrrol-3-yl H H Br 2 CH₂CF₃ S Pyrrol-4-yl I H I 2 CH₂CH(CH₃)₂ SPyrrol-5-yl H I H 2 CH₂C(CH₃)₃ S Thiophene-2-yl H H I 2 H₂C—C≡N SThiophene-3-yl CH₃ H CH₃ 2 H₂C—C≡CH S Thiophene-4-yl H CH₃ H 2 CH(CH₃)₂S Thiophene-5-yl H H CH₃ 3 CH₂CH₂OH S Oxazol-2-yl C₂H₅ H C₂H₅ 3CH₂CH(CH₃)OH S Oxazol-4-yl H C₂H₅ H 3 CH(CH₃)CH₂OH S Oxazol-5-yl H HC₂H₅ 3 CH(CH₃)CH(CH₃)OH S Isoxazol-3-yl i-C₃H₇ H i-C₃H₇ 3 C(CH₃)₂CH₂OH SIsoxazol-4-yl H i-C₃H₇ H 3 CH₂C(CH₃)₂OH S Isoxazol-5-yl H H i-C₃H₇ 3CH₂CHF₂ O Pyrazol-3-yl Cl H Cl 3 CH₂CF₃ O Pyrazol-4-yl H Cl H 3CH₂CH(CH₃)₂ O Pyrazol-5-yl H H Cl 3 CH₂C(CH₃)₃ O Thiazol-2-yl Br H Br 3H₂C—C≡N O Thiazol-4-yl H Br H 3 H₂C—C≡CH O Thiazol-5-yl H H Br 2CH(CH₃)₂ O Furan-2-yl I H I 2 CH₂CH₂OH O Furan-3-yl H I H 2 CH₂CH(CH₃)OHO Furan-4-yl H H I 2 CH(CH₃)CH₂OH O Furan-5-yl CH₃ H CH₃ 2CH(CH₃)CH(CH₃)OH O Pyrrol-2-yl H CH₃ H 2 C(CH₃)₂CH₂OH O Pyrrol-3-yl H HCH₃ 2 CH₂C(CH₃)₂OH O Pyrrol-4-yl C₂H₅ H C₂H₅ 2 CH₂CHF₂ O Pyrrol-5-yl HC₂H₅ H 2 CH₂CF₃ O Thiophene-2-yl H H C₂H₅ 2 CH₂CH(CH₃)₂ O Thiophene-3-yli-C₃H₇ H i-C₃H₇ 2 CH₂C(CH₃)₃ O Thiophene-4-yl H i-C₃H₇ H 2 H₂C—C≡N OThiophene-5-yl H H i-C₃H₇ 3 H₂C—C≡CH S═O Oxazol-2-yl Cl H Cl 3 CH(CH₃)₂S═O Oxazol-4-yl H Cl H 3 CH₂CH₂OH S═O Oxazol-5-yl H H Cl 3 CH₂CH(CH₃)OHS═O Isoxazol-3-yl Br H Br 3 CH(CH₃)CH₂OH S═O Isoxazol-4-yl H Br H 3CH(CH₃)CH(CH₃)OH S═O Isoxazol-5-yl H H Br 3 C(CH₃)₂CH₂OH S═OPyrazol-3-yl I H I 3 CH₂C(CH₃)₂OH S═O Pyrazol-4-yl H I H 3 CH₂CHF₂ S═OPyrazol-5-yl H H I 3 CH₂CF₃ S═O Thiazol-2-yl CH₃ H CH₃ 3 CH₂CH(CH₃)₂ S═OThiazol-4-yl H CH₃ H 3 CH₂C(CH₃)₃ S═O Thiazol-5-yl H H CH₃ 2 H₂C—C≡N S═OFuran-2-yl C₂H₅ H C₂H₅ 2 H₂C—C≡CH S═O Furan-3-yl H C₂H₅ H 2 CH(CH₃)₂ S═OFuran-4-yl H H C₂H₅ 2 CH₂CH₂OH S═O Furan-5-yl i-C₃H₇ H i-C₃H₇ 2CH₂CH(CH₃)OH S═O Pyrrol-2-yl H H i-C₃H₇ 2 CH(CH₃)CH₂OH S═O Pyrrol-3-yl HH i-C₃H₇ 2 CH(CH₃)CH(CH₃)OH O═S═O Pyrrol-4-yl Cl H Cl 2 C(CH₃)₂CH₂OHO═S═O Pyrrol-5-yl H Cl H 2 CH₂C(CH₃)₂OH O═S═O Thiophene-2-yl H H Cl 2CH₂CHF₂ O═S═O Thiophene-3-yl Br H Br 2 CH₂CF₃ O═S═O Thiophene-4-yl H BrH 2 CH₂CH(CH₃)₂ O═S═O Thiophene-5-yl H H Br 3 CH₂C(CH₃)₃ O═S═OOxazol-2-yl I H I 3 H₂C—C≡N O═S═O Oxazol-4-yl H I H 3 H₂C—C≡CH O═S═OOxazol-5-yl H H I 3 CH(CH₃)₂ O═S═O Isoxazol-3-yl CH₃ H CH₃ 3 CH₂CH₂OHO═S═O Isoxazol-4-yl H CH₃ H 3 CH₂CH(CH₃)OH O═S═O Isoxazol-5-yl H H CH₃ 3CH(CH₃)CH₂OH O═S═O Pyrazol-3-yl C₂H₅ H C₂H₅ 3 CH(CH₃)CH(CH₃)OH O═S═OPyrazol-4-yl H C₂H₅ H 3 C(CH₃)₂CH₂OH O═S═O Pyrazol-5-yl H H C₂H₅ 3CH₂C(CH₃)₂OH O═S═O Thiazol-2-yl i-C₃H₇ H i-C₃H₇ 3 CH₂CHF₂ O═S═OThiazol-4-yl H H i-C₃H₇ 3 CH₂CF₃ O═S═O Thiazol-5-yl H H i-C₃H₇ 2CH₂CH(CH₃)₂ NH Furan-2-yl Cl H Cl 2 CH₂C(CH₃)₃ NH Furan-3-yl H Cl H 2H₂C—C≡N NH Furan-4-yl H H Cl 2 H₂C—C≡CH NH Furan-5-yl Br H Br 2 CH(CH₃)₂NH Pyrrol-2-yl H Br H 2 CH₂CH₂OH NH Pyrrol-3-yl H H Br 2 CH₂CH(CH₃)OH NHPyrrol-4-yl I H I 2 CH(CH₃)CH₂OH NH Pyrrol-5-yl H I H 2 CH(CH₃)CH(CH₃)OHNH Thiophene-2-yl H H I 2 C(CH₃)₂CH₂OH NH Thiophene-3-yl CH₃ H CH₃ 2CH₂C(CH₃)₂OH NH Thiophene-4-yl H CH₃ H 2 CH₂CHF₂ NH Thiophene-5-yl H HCH₃ 3 CH₂CF₃ NH Oxazol-2-yl C₂H₅ H C₂H₅ 3 CH₂CH(CH₃)₂ NH Oxazol-4-yl HC₂H₅ H 3 CH₂C(CH₃)₃ NH Oxazol-5-yl H H C₂H₅ 3 H₂C—C≡N NH Isoxazol-3-yli-C₃H₇ H i-C₃H₇ 3 H₂C—C≡CH NH Isoxazol-4-yl H H i-C₃H₇ 3 CH(CH₃)₂ NHIsoxazol-5-yl H H i-C₃H₇ 3 CH₂CH₂OH C═O Pyrazol-3-yl Cl H Cl 3CH₂CH(CH₃)OH C═O Pyrazol-4-yl H Cl H 3 CH(CH₃)CH₂OH C═O Pyrazol-5-yl H HCl 3 CH(CH₃)CH(CH₃)OH C═O Thiazol-2-yl Br H Br 3 C(CH₃)₂CH₂OH C═OThiazol-4-yl H Br H 3 CH₂C(CH₃)₂OH C═O Thiazol-5-yl H H Br 3 CH₂CHF₂ C═OFuran-2-yl I H I 2 CH₂CF₃ C═O Furan-3-yl H I H 2 CH₂CH(CH₃)₂ C═OFuran-4-yl H H I 2 CH₂C(CH₃)₃ C═O Furan-5-yl CH₃ H CH₃ 2 H₂C—C≡N C═OPyrrol-2-yl H CH₃ H 2 H₂C—C≡CH C═O Pyrrol-3-yl H H CH₃ 2 CH(CH₃)₂ C═OPyrrol-4-yl C₂H₅ H C₂H₅ 2 CH₂CH₂OH C═O Pyrrol-5-yl H C₂H₅ H 2CH₂CH(CH₃)OH C═O Thiophene-2-yl H H C₂H₅ 2 CH(CH₃)CH₂OH C═OThiophene-3-yl i-C₃H₇ H i-C₃H₇ 2 CH(CH₃)CH(CH₃)OH C═O Thiophene-4-yl Hi-C₃H₇ H 2 C(CH₃)₂CH₂OH C═O Thiophene-5-yl H H i-C₃H₇ 3 CH₂C(CH₃)₂OH C═SOxazol-2-yl Cl H Cl 3 CH₂CHF₂ C═S Oxazol-4-yl H Cl H 3 CH₂CF₃ C═SOxazol-5-yl H H Cl 3 CH₂CH(CH₃)₂ C═S Isoxazol-3-yl Br H Br 3 CH₂C(CH₃)₃C═S Isoxazol-4-yl H Br H 3 H₂C—C≡N C═S Isoxazol-5-yl H H Br 3 H₂C—C≡CHC═S Pyrazol-3-yl I H I 3 CH(CH₃)₂ C═S Pyrazol-4-yl H I H 3 CH₂CH₂OH C═SPyrazol-5-yl H H I 3 CH₂CH(CH₃)OH C═S Thiazol-2-yl CH₃ H CH₃ 2CH(CH₃)CH₂OH C═S Thiazol-4-yl H CH₃ H 2 CH(CH₃)CH(CH₃)OH C═SThiazol-5-yl H H CH₃ 2 C(CH₃)₂CH₂OH C═S Furan-2-yl C₂H₅ H C₂H₅ 2CH₂C(CH₃)₂OH C═S Furan-3-yl H C₂H₅ H 2 CH₂CHF₂ C═S Furan-4-yl H H C₂H₅ 2CH₂CF₃ C═S Furan-5-yl i-C₃H₇ H i-C₃H₇ 2 CH₂CH(CH₃)₂ C═S Pyrrol-2-yl Hi-C₃H₇ H 2 CH₂C(CH₃)₃ C═S Pyrrol-3-yl H H i-C₃H₇ 3 H₂C—C≡N CH₂Pyrrol-4-yl Cl H Cl 3 H₂C—C≡CH CH₂ Pyrrol-5-yl H Cl H 3 CH(CH₃)₂ CH₂Thiophene-2-yl H H Cl 3 CH₂CH₂OH CH₂ Thiophene-3-yl Br H Br 3CH₂CH(CH₃)OH CH₂ Thiophene-4-yl H Br H 3 CH(CH₃)CH₂OH CH₂ Thiophene-5-ylH H Br 2 CH(CH₃)CH(CH₃)OH CH₂ Oxazol-2-yl I H I 2 C(CH₃)₂CH₂OH CH₂Oxazol-4-yl H I H 2 CH₂C(CH₃)₂OH CH₂ Oxazol-5-yl H H I 2 CH₂CHF₂ CH₂Isoxazol-3-yl CH₃ H CH₃ 2 CH₂CF₃ CH₂ Isoxazol-4-yl H CH₃ H 2 CH₂CH(CH₃)₂CH₂ Isoxazol-5-yl H H CH₃ 3 CH₂C(CH₃)₃ CH₂ Pyrazol-3-yl C₂H₅ H C₂H₅ 3H₂C—C≡N CH₂ Pyrazol-4-yl H C₂H₅ H 3 H₂C—C≡CH CH₂ Pyrazol-5-yl H H C₂H₅ 3CH(CH₃)₂ CH₂ Thiazol-2-yl i-C₃H₇ H i-C₃H₇ 3 CH₂CH₂OH CH₂ Thiazol-4-yl Hi-C₃H₇ H 3 CH₂CH(CH₃)OH CH₂ Thiazol-5-yl H H i-C₃H₇ 2 CH(CH₃)CH₂OH CH—OHFuran-2-yl Cl H Cl 2 CH(CH₃)CH(CH₃)OH CH—OH Furan-3-yl H Cl H 2C(CH₃)₂CH₂OH CH—OH Furan-4-yl H H Cl 2 CH₂C(CH₃)₂OH CH—OH Furan-5-yl BrH Br 2 CH₂CHF₂ CH—OH Pyrrol-2-yl H Br H 2 CH₂CF₃ CH—OH Pyrrol-3-yl H HBr 3 CH₂CH(CH₃)₂ CH—OH Pyrrol-4-yl I H I 3 CH₂C(CH₃)₃ CH—OH Pyrrol-5-ylH I H 3 H₂C—C≡N CH—OH Thiophene-2-yl H H I 3 H₂C—C≡CH CH—OHThiophene-3-yl CH₃ H CH₃ 3 CH(CH₃)₂ CH—OH Thiophene-4-yl H CH₃ H 3CH₂CH₂OH CH—OH Thiophene-5-yl H H CH₃ 2 CH₂CH(CH₃)OH CH—OH Oxazol-2-ylC₂H₅ H C₂H₅ 2 CH(CH₃)CH₂OH CH—OH Oxazol-4-yl H C₂H₅ H 2 CH(CH₃)CH(CH₃)OHCH—OH Oxazol-5-yl H H C₂H₅ 2 C(CH₃)₂CH₂OH CH—OH Isoxazol-3-yl i-C₃H₇ Hi-C₃H₇ 2 CH₂C(CH₃)₂OH CH—OH Isoxazol-4-yl H i-C₃H₇ H 2 CH₂CHF₂ CH—OHIsoxazol-5-yl H H i-C₃H₇ 3 CH₂CF₃ CH—F Pyrazol-3-yl Cl H Cl 3CH₂CH(CH₃)₂ CH—F Pyrazol-4-yl H Cl H 3 CH₂C(CH₃)₃ CH—F Pyrazol-5-yl H HCl 3 H₂C—C≡N CH—F Thiazol-2-yl Br H Br 3 H₂C—C≡CH CH—F Thiazol-4-yl H BrH 3 CH(CH₃)₂ CH—F Thiazol-5-yl H H Br 2 CH₂CH₂OH CH—F Furan-2-yl I H I 2CH₂CH(CH₃)OH CH—F Furan-3-yl H I H 2 CH(CH₃)CH₂OH CH—F Furan-4-yl H H I2 CH(CH₃)CH(CH₃)OH CH—F Furan-5-yl CH₃ H CH₃ 2 C(CH₃)₂CH₂OH CH—FPyrrol-2-yl H CH₃ H 2 CH₂C(CH₃)₂OH CH—F Pyrrol-3-yl H H CH₃ 3 CH₂CHF₂CH—F Pyrrol-4-yl C₂H₅ H C₂H₅ 3 CH₂CF₃ CH—F Pyrrol-5-yl H C₂H₅ H 3CH₂CH(CH₃)₂ CH—F Thiophene-2-yl H H C₂H₅ 3 CH₂C(CH₃)₃ CH—FThiophene-3-yl i-C₃H₇ H i-C₃H₇ 3 H₂C—C≡N CH—F Thiophene-4-yl H i-C₃H₇ H3 H₂C—C≡CH CH—F Thiophene-5-yl H H i-C₃H₇ 2 CH(CH₃)₂ S Oxazol-2-yl Cl ClCl 2 CH₂CH₂OH S Oxazol-4-yl Cl H Cl 2 CH₂CH(CH₃)OH S Oxazol-5-yl Br BrBr 2 CH(CH₃)CH₂OH S Isoxazol-3-yl Br H Br 2 CH(CH₃)CH(CH₃)OH SIsoxazol-4-yl I I I 2 C(CH₃)₂CH₂OH S Isoxazol-5-yl I H I 3 CH₂C(CH₃)₂OHS Pyrazol-3-yl CH₃ CH₃ CH₃ 3 CH₂CHF₂ S Pyrazol-4-yl CH₃ H CH₃ 3 CH₂CF₃ SPyrazol-5-yl C₂H₅ C₂H₅ C₂H₅ 3 CH₂CH(CH₃)₂ S Thiazol-2-yl C₂H₅ H C₂H₅ 3CH₂C(CH₃)₃ S Thiazol-4-yl i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 H₂C—C≡N S Thiazol-5-yli-C₃H₇ H i-C₃H₇ 2 H₂C—C≡CH O Furan-2-yl Cl Cl Cl 2 CH(CH₃)₂ O Furan-3-ylCl H Cl 2 CH₂CH₂OH O Furan-4-yl Br Br Br 2 CH₂CH(CH₃)OH O Furan-5-yl BrH Br 2 CH(CH₃)CH₂OH O Pyrrol-2-yl I I I 2 CH(CH₃)CH(CH₃)OH O Pyrrol-3-ylI H I 3 C(CH₃)₂CH₂OH O Pyrrol-4-yl CH₃ CH₃ CH₃ 3 CH₂C(CH₃)₂OH OPyrrol-5-yl CH₃ H CH₃ 3 CH₂CHF₂ O Thiophene-2-yl C₂H₅ C₂H₅ C₂H₅ 3 CH₂CF₃O Thiophene-3-yl C₂H₅ H C₂H₅ 3 CH₂CH(CH₃)₂ O Thiophene-4-yl i-C₃H₇i-C₃H₇ i-C₃H₇ 3 CH₂C(CH₃)₃ O Thiophene-5-yl i-C₃H₇ H i-C₃H₇ 2 H₂C—C≡NS═O Oxazol-2-yl Cl Cl Cl 2 H₂C—C≡CH S═O Oxazol-4-yl Cl H Cl 2 CH(CH₃)₂S═O Oxazol-5-yl Br Br Br 2 CH₂CH₂OH S═O Isoxazol-3-yl Br H Br 2CH₂CH(CH₃)OH S═O Isoxazol-4-yl I I I 2 CH(CH₃)CH₂OH S═O Isoxazol-5-yl IH I 3 CH(CH₃)CH(CH₃)OH S═O Pyrazol-3-yl CH₃ CH₃ CH₃ 3 C(CH₃)₂CH₂OH S═OPyrazol-4-yl CH₃ H CH₃ 3 CH₂C(CH₃)₂OH S═O Pyrazol-5-yl C₂H₅ C₂H₅ C₂H₅ 3CH₂CHF₂ S═O Thiazol-2-yl C₂H₅ H C₂H₅ 3 CH₂CF₃ S═O Thiazol-4-yl i-C₃H₇i-C₃H₇ i-C₃H₇ 3 CH₂CH(CH₃)₂ S═O Thiazol-5-yl i-C₃H₇ H i-C₃H₇ 2CH₂C(CH₃)₃ O═S═O Furan-2-yl Cl Cl Cl 2 H₂C—C≡N O═S═O Furan-3-yl Cl H Cl2 H₂C—C≡CH O═S═O Furan-4-yl Br Br Br 2 CH(CH₃)₂ O═S═O Furan-5-yl Br H Br2 CH₂CH₂OH O═S═O Pyrrol-2-yl I I I 2 CH₂CH(CH₃)OH O═S═O Pyrrol-3-yl I HI 3 CH(CH₃)CH₂OH O═S═O Pyrrol-4-yl CH₃ CH₃ CH₃ 3 CH(CH₃)CH(CH₃)OH O═S═OPyrrol-5-yl CH₃ H CH₃ 3 C(CH₃)₂CH₂OH O═S═O Thiophene-2-yl C₂H₅ C₂H₅ C₂H₅3 CH₂C(CH₃)₂OH O═S═O Thiophene-3-yl C₂H₅ H C₂H₅ 3 CH₂CHF₂ O═S═OThiophene-4-yl i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH₂CF₃ O═S═O Thiophene-5-yl i-C₃H₇H i-C₃H₇ 2 CH₂CH(CH₃)₂ NH Oxazol-2-yl Cl Cl Cl 2 CH₂C(CH₃)₃ NHOxazol-4-yl Cl H Cl 2 H₂C—C≡N NH Oxazol-5-yl Br Br Br 2 H₂C—C≡CH NHIsoxazol-3-yl Br H Br 2 CH(CH₃)₂ NH Isoxazol-4-yl I I I 2 CH₂CH₂OH NHIsoxazol-5-yl I H I 3 CH₂CH(CH₃)OH NH Pyrazol-3-yl CH₃ CH₃ CH₃ 3CH(CH₃)CH₂OH NH Pyrazol-4-yl CH₃ H CH₃ 3 CH(CH₃)CH(CH₃)OH NHPyrazol-5-yl C₂H₅ C₂H₅ C₂H₅ 3 C(CH₃)₂CH₂OH NH Thiazol-2-yl C₂H₅ H C₂H₅ 3CH₂C(CH₃)₂OH NH Thiazol-4-yl i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH₂CHF₂ NHThiazol-5-yl i-C₃H₇ H i-C₃H₇ 2 CH₂CF₃ C═O Furan-2-yl Cl Cl Cl 2CH₂CH(CH₃)₂ C═O Furan-3-yl Cl H Cl 2 CH₂C(CH₃)₃ C═O Furan-4-yl Br Br Br2 H₂C—C≡N C═O Furan-5-yl Br H Br 2 H₂C—C≡CH C═O Pyrrol-2-yl I I I 3CH(CH₃)₂ C═O Pyrrol-3-yl I H I 3 CH₂CH₂OH C═O Pyrrol-4-yl CH₃ CH₃ CH₃ 3CH₂CH(CH₃)OH C═O Pyrrol-5-yl CH₃ H CH₃ 3 CH(CH₃)CH₂OH C═O Thiophene-2-ylC₂H₅ C₂H₅ C₂H₅ 3 CH(CH₃)CH(CH₃)OH C═O Thiophene-3-yl C₂H₅ H C₂H₅ 3C(CH₃)₂CH₂OH C═O Thiophene-4-yl i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH₂C(CH₃)₂OH C═OThiophene-5-yl i-C₃H₇ H i-C₃H₇ 2 CH₂CHF₂ C═S Oxazol-2-yl Cl Cl Cl 2CH₂CF₃ C═S Oxazol-4-yl Cl H Cl 2 CH₂CH(CH₃)₂ C═S Oxazol-5-yl Br Br Br 2CH₂C(CH₃)₃ C═S Isoxazol-3-yl Br H Br 2 H₂C—C≡N C═S Isoxazol-4-yl I I I 2H₂C—C≡CH C═S Isoxazol-5-yl I H I 3 CH(CH₃)₂ C═S Pyrazol-3-yl CH₃ CH₃ CH₃3 CH₂CH₂OH C═S Pyrazol-4-yl CH₃ H CH₃ 3 CH₂CH(CH₃)OH C═S Pyrazol-5-ylC₂H₅ C₂H₅ C₂H₅ 3 CH(CH₃)CH₂OH C═S Thiazol-2-yl C₂H₅ H C₂H₅ 3CH(CH₃)CH(CH₃)OH C═S Thiazol-4-yl i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 C(CH₃)₂CH₂OHC═S Thiazol-5-yl i-C₃H₇ H i-C₃H₇ 2 CH₂C(CH₃)₂OH CH₂ Furan-2-yl Cl Cl Cl2 CH₂CHF₂ CH₂ Furan-3-yl Cl H Cl 2 CH₂CF₃ CH₂ Furan-4-yl Br Br Br 2CH₂CH(CH₃)₂ CH₂ Furan-5-yl Br H Br 2 CH₂C(CH₃)₃ CH₂ Pyrrol-2-yl I I I 2H₂C—C≡N CH₂ Pyrrol-3-yl I H I 3 H₂C—C≡CH CH₂ Pyrrol-4-yl CH₃ CH₃ CH₃ 3CH(CH₃)₂ CH₂ Pyrrol-5-yl CH₃ H CH₃ 3 CH₂CH₂OH CH₂ Thiophene-2-yl C₂H₅C₂H₅ C₂H₅ 3 CH₂CH(CH₃)OH CH₂ Thiophene-3-yl C₂H₅ H C₂H₅ 3 CH(CH₃)CH₂OHCH₂ Thiophene-4-yl i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH(CH₃)CH(CH₃)OH CH₂Thiophene-5-yl i-C₃H₇ H i-C₃H₇ 2 C(CH₃)₂CH₂OH CH—OH Oxazol-2-yl Cl Cl Cl2 CH₂C(CH₃)₂OH CH—OH Oxazol-4-yl Cl H Cl 2 CH₂CHF₂ CH—OH Oxazol-5-yl BrBr Br 2 CH₂CF₃ CH—OH Isoxazol-3-yl Br H Br 2 CH₂CH(CH₃)₂ CH—OHIsoxazol-4-yl I I I 2 CH₂C(CH₃)₃ CH—OH Isoxazol-5-yl I H I 3 H₂C—C≡NCH—OH Pyrazol-3-yl CH₃ CH₃ CH₃ 3 H₂C—C≡CH CH—OH Pyrazol-4-yl CH₃ H CH₃ 3CH(CH₃)₂ CH—OH Pyrazol-5-yl C₂H₅ C₂H₅ C₂H₅ 3 CH₂CH₂OH CH—OH Thiazol-2-ylC₂H₅ H C₂H₅ 3 CH₂CH(CH₃)OH CH—OH Thiazol-4-yl i-C₃H₇ i-C₃H₇ i-C₃H₇ 3CH(CH₃)CH₂OH CH—OH Thiazol-5-yl i-C₃H₇ H i-C₃H₇ 2 CH(CH₃)CH(CH₃)OH CH—FFuran-2-yl Cl Cl Cl 2 C(CH₃)₂CH₂OH CH—F Furan-3-yl Cl H Cl 2CH₂C(CH₃)₂OH CH—F Furan-4-yl Br Br Br 2 CH₂CHF₂ CH—F Furan-5-yl Br H Br2 CH₂CF₃ CH—F Pyrrol-2-yl I I I 2 CH₂CH(CH₃)₂ CH—F Pyrrol-3-yl I H I 3CH₂C(CH₃)₃ CH—F Pyrrol-4-yl CH₃ CH₃ CH₃ 3 H₂C—C≡N CH—F Pyrrol-5-yl CH₃ HCH₃ 3 H₂C—C≡CH CH—F Thiophene-2-yl C₂H₅ C₂H₅ C₂H₅ 3 CH(CH₃)₂ CH—FThiophene-3-yl C₂H₅ H C₂H₅ 3 CH₂CH₂OH CH—F Thiophene-4-yl i-C₃H₇ i-C₃H₇i-C₃H₇ 3 CH₂CH(CH₃)OH CH—F Thiophene-5-yl i-C₃H₇ H i-C₃H₇ 2 CH(CH₃)CH₂OHS Oxazol-2-yl H Cl H 2 CH(CH₃)CH(CH₃)OH S═O Oxazol-4-yl H H Cl 2C(CH₃)₂CH₂OH O═S═O Oxazol-5-yl H Cl H 2 CH₂C(CH₃)₂OH CH₂ Isoxazol-3-yl HH Cl 2 CH₂CHF₂ C═O Isoxazol-4-yl H I H 3 CH₂CF₃ C═S Isoxazol-5-yl H H I3 CH₂CH(CH₃)₂ CH—OH Pyrazol-3-yl H Br H 3 CH₂C(CH₃)₃ CH—F Pyrazol-4-yl HH Br 3 H₂C—C≡N O Pyrazol-5-yl Cl H Cl 3 H₂C—C≡CH NH Thiazol-2-yl Cl H Cl

TABLE 3

m Y X² X³ X⁴ X⁵ 2 S H Cl H H 2 S H H H Cl 2 S H H Cl H 2 S Cl H H H 2 SH Br H H 2 S H H H Br 3 S H H Br H 3 S Br H H H 3 S H I H H 3 S H H H I3 S H H I H 3 S I H H H 4 S H CH₃ H H 4 S H H H CH₃ 4 S H H CH₃ H 4 SCH₃ H H H 4 S H C₂H₅ H H 4 S H H H C₂H₅ 5 S H H C₂H₅ H 5 S C₂H₅ H H H 5S H i-C₃H₇ H H 5 S H H H i-C₃H₇ 5 S H H i-C₃H₇ H 5 S i-C₃H₇ H H H 2 O HCl H H 2 O H H H Cl 2 O H H Cl H 2 O Cl H H H 2 O H Br H H 2 O H H H Br3 O H H Br H 3 O Br H H H 3 O H I H H 3 O H H H I 3 O H H I H 3 O I H HH 4 O H CH₃ H H 4 O H H H CH₃ 4 O H H CH₃ H 4 O CH₃ H H H 4 O H C₂H₅ H H4 O H H H C₂H₅ 5 O H H C₂H₅ H 5 O C₂H₅ H H H 5 O H i-C₃H₇ H H 5 O H H Hi-C₃H₇ 5 O H H i-C₃H₇ H 5 O i-C₃H₇ H H H 2 S═O H Cl H H 2 S═O H H H Cl 2S═O H H Cl H 2 S═O Cl H H H 2 S═O H Br H H 2 S═O H H H Br 3 S═O H H Br H3 S═O Br H H H 3 S═O H I H H 3 S═O H H H I 3 S═O H H I H 3 S═O I H H H 4S═O H CH₃ H H 4 S═O H H H CH₃ 4 S═O H H CH₃ H 4 S═O CH₃ H H H 4 S═O HC₂H₅ H H 4 S═O H H H C₂H₅ 5 S═O H H C₂H₅ H 5 S═O C₂H₅ H H H 5 S═O H H Hi-C₃H₇ 5 S═O H H i-C₃H₇ H 5 S═O i-C₃H₇ H H H 5 S═O H H H i-C₃H₇ 2 O═S═OH Cl H H 2 O═S═O H H H Cl 2 O═S═O H H Cl H 2 O═S═O Cl H H H 2 O═S═O H BrH H 2 O═S═O H H H Br 3 O═S═O H H Br H 3 O═S═O Br H H H 3 O═S═O H I H H 3O═S═O H H H I 3 O═S═O H H I H 3 O═S═O I H H H 4 O═S═O H CH₃ H H 4 O═S═OH H H CH₃ 4 O═S═O H H CH₃ H 4 O═S═O CH₃ H H H 4 O═S═O H C₂H₅ H H 4 O═S═OH H H C₂H₅ 5 O═S═O H H C₂H₅ H 5 O═S═O C₂H₅ H H H 5 O═S═O H i-C₃H₇ H H 5O═S═O H H H i-C₃H₇ 5 O═S═O H H i-C₃H₇ H 5 O═S═O i-C₃H₇ H H H 2 NH H Cl HH 2 NH H H H Cl 2 NH H H Cl H 2 NH Cl H H H 2 NH H Br H H 2 NH H H H Br3 NH H H Br H 3 NH Br H H H 3 NH H I H H 3 NH H H H I 3 NH H H I H 3 NHI H H H 4 NH H CH₃ H H 4 NH H H H CH₃ 4 NH H H CH₃ H 4 NH CH₃ H H H 4 NHH C₂H₅ H H 4 NH H H H C₂H₅ 5 NH H H C₂H₅ H 5 NH C₂H₅ H H H 5 NH H i-C₃H₇H H 5 NH H H H i-C₃H₇ 5 NH H H i-C₃H₇ H 5 NH i-C₃H₇ H H H 2 C═O H Cl H H2 C═O H H H Cl 2 C═O H H Cl H 2 C═O Cl H H H 2 C═O H Br H H 2 C═O H H HBr 3 C═O H H Br H 3 C═O Br H H H 3 C═O H I H H 3 C═O H H H I 3 C═O H H IH 3 C═O I H H H 4 C═O H CH₃ H H 4 C═O H H H CH₃ 4 C═O H H CH₃ H 4 C═OCH₃ H H H 4 C═O H C₂H₅ H H 4 C═O H H H C₂H₅ 5 C═O H H C₂H₅ H 5 C═O C₂H₅H H H 5 C═O H i-C₃H₇ H H 5 C═O H H H i-C₃H₇ 5 C═O H H i-C₃H₇ H 5 C═Oi-C₃H₇ H H H 2 C═S H Cl H H 2 C═S H H H Cl 2 C═S H H Cl H 2 C═S Cl H H H2 C═S H Br H H 2 C═S H H H Br 3 C═S H H Br H 3 C═S Br H H H 3 C═S H I HH 3 C═S H H H I 3 C═S H H I H 3 C═S I H H H 4 C═S H CH₃ H H 4 C═S H H HCH₃ 4 C═S H H CH₃ H 4 C═S CH₃ H H H 4 C═S H C₂H₅ H H 4 C═S H H H C₂H₅ 5C═S H H C₂H₅ H 5 C═S C₂H₅ H H H 5 C═S H i-C₃H₇ H H 5 C═S H H H i-C₃H₇ 5C═S H H i-C₃H₇ H 5 C═S i-C₃H₇ H H H 2 CH₂ H Cl H H 2 CH₂ H H H Cl 2 CH₂H H Cl H 2 CH₂ Cl H H H 2 CH₂ H Br H H 2 CH₂ H H H Br 3 CH₂ H H Br H 3CH₂ Br H H H 3 CH₂ H I H H 3 CH₂ H H H I 3 CH₂ H H I H 3 CH₂ I H H H 4CH₂ H CH₃ H H 4 CH₂ H H H CH₃ 4 CH₂ H H CH₃ H 4 CH₂ CH₃ H H H 4 CH₂ HC₂H₅ H H 4 CH₂ H H H C₂H₅ 5 CH₂ H H C₂H₅ H 5 CH₂ C₂H₅ H H H 5 CH₂ Hi-C₃H₇ H H 5 CH₂ H H H i-C₃H₇ 5 CH₂ H H i-C₃H₇ H 5 CH₂ i-C₃H₇ H H H 2CH—OH H Cl H H 2 CH—OH H H H Cl 2 CH—OH H H Cl H 2 CH—OH Cl H H H 2CH—OH H Br H H 2 CH—OH H H H Br 3 CH—OH H H Br H 3 CH—OH Br H H H 3CH—OH H I H H 3 CH—OH H H H I 3 CH—OH H H I H 3 CH—OH I H H H 4 CH—OH HCH₃ H H 4 CH—OH H H H CH₃ 4 CH—OH H H CH₃ H 4 CH—OH CH₃ H H H 4 CH—OH HC₂H₅ H H 4 CH—OH H H H C₂H₅ 5 CH—OH H H C₂H₅ H 5 CH—OH C₂H₅ H H H 5CH—OH H i-C₃H₇ H H 5 CH—OH H H H i-C₃H₇ 5 CH—OH H H i-C₃H₇ H 5 CH—OHi-C₃H₇ H H H 2 CH—F H Cl H H 2 CH—F H H H Cl 2 CH—F H H Cl H 2 CH—F Cl HH H 2 CH—F H Br H H 2 CH—F H H H Br 3 CH—F H H Br H 3 CH—F Br H H H 3CH—F H I H H 3 CH—F H H H I 3 CH—F H H I H 3 CH—F I H H H 4 CH—F H CH₃ HH 4 CH—F H H H CH₃ 4 CH—F H H CH₃ H 4 CH—F CH₃ H H H 4 CH—F H C₂H₅ H H 4CH—F H H H C₂H₅ 5 CH—F H H C₂H₅ H 5 CH—F C₂H₅ H H H 5 CH—F H i-C₃H₇ H H5 CH—F H H H i-C₃H₇ 5 CH—F H H i-C₃H₇ H 5 CH—F i-C₃H₇ H H H 2 S H Cl HCl 2 S Cl H Cl H 2 S Cl H H Cl 2 S H Br H Br 2 S Br H Br H 2 S Br H H Br3 S H I H I 3 S I H I H 3 S I H H I 3 S H CH₃ H CH₃ 3 S CH₃ H CH₃ H 3 SCH₃ H H CH₃ 4 S H C₂H₅ H C₂H₅ 4 S C₂H₅ H C₂H₅ H 4 S C₂H₅ H H C₂H₅ 4 S Hi-C₃H₇ H i-C₃H₇ 4 S i-C₃H₇ H i-C₃H₇ H 4 S i-C₃H₇ H H i-C₃H₇ 5 O H Cl HCl 5 O Cl H Cl H 5 O Cl H H Cl 5 O H Br H Br 5 O Br H Br H 5 O Br H H Br2 O H I H I 2 O I H I H 2 O I H H I 2 O H CH₃ H CH₃ 2 O CH₃ H CH₃ H 2 OCH₃ H H CH₃ 3 O H C₂H₅ H C₂H₅ 3 O C₂H₅ H C₂H₅ H 3 O C₂H₅ H H C₂H₅ 3 O Hi-C₃H₇ H i-C₃H₇ 3 O i-C₃H₇ H i-C₃H₇ H 3 O i-C₃H₇ H H i-C₃H₇ 4 S═O H Cl HCl 4 S═O Cl H Cl H 4 S═O Cl H H Cl 4 S═O H Br H Br 4 S═O Br H Br H 4 S═OBr H H Br 5 S═O H I H I 5 S═O I H I H 5 S═O I H H I 5 S═O H CH₃ H CH₃ 5S═O CH₃ H CH₃ H 5 S═O CH H H CH₃ 2 S═O H C₂H₅ H C₂H₅ 2 S═O C₂H₅ H C₂H₅ H2 S═O C₂H₅ H H C₂H₅ 2 S═O H i-C₃H₇ H i-C₃H₇ 2 S═O i-C₃H₇ H i-C₃H₇ H 2S═O i-C₃H₇ H H i-C₃H₇ 3 O═S═O H Cl H Cl 3 O═S═O Cl H Cl H 3 O═S═O Cl H HCl 3 O═S═O H Br H Br 3 O═S═O Br H Br H 3 O═S═O Br H H Br 4 O═S═O H I H I4 O═S═O I H I H 4 O═S═O I H H I 4 O═S═O H CH₃ H CH₃ 4 O═S═O CH₃ H CH₃ H4 O═S═O CH₃ H H CH₃ 5 O═S═O H C₂H₅ H C₂H₅ 5 O═S═O C₂H₅ H C₂H₅ H 5 O═S═OC₂H₅ H H C₂H₅ 5 O═S═O H i-C₃H₇ H i-C₃H₇ 5 O═S═O i-C₃H₇ H i-C₃H₇ H 5O═S═O i-C₃H₇ H H i-C₃H₇ 2 NH H Cl H Cl 2 NH Cl H Cl H 2 NH Cl H H Cl 2NH H Br H Br 2 NH Br H Br H 2 NH Br H H Br 2 NH H I H I 3 NH I H I H 3NH I H H I 3 NH H CH₃ H CH₃ 3 NH CH₃ H CH₃ H 3 NH CH₃ H H CH₃ 3 NH HC₂H₅ H C₂H₅ 4 NH C₂H₅ H C₂H₅ H 4 NH C₂H₅ H H C₂H₅ 4 NH H i-C₃H₇ H i-C₃H₇4 NH i-C₃H₇ H i-C₃H₇ H 4 NH i-C₃H₇ H H i-C₃H₇ 4 C═O H Cl H Cl 5 C═O Cl HCl H 5 C═O Cl H H Cl 5 C═O H Br H Br 5 C═O Br H Br H 5 C═O Br H H Br 2C═O H I H I 2 C═O I H I H 2 C═O I H H I 2 C═O H CH₃ H CH₃ 2 C═O CH₃ HCH₃ H 2 C═O CH₃ H H CH₃ 3 C═O H C₂H₅ H C₂H₅ 3 C═O C₂H₅ H C₂H₅ H 3 C═OC₂H₅ H H C₂H₅ 3 C═O H i-C₃H₇ H i-C₃H₇ 3 C═O i-C₃H₇ H i-C₃H₇ H 3 C═Oi-C₃H₇ H H i-C₃H₇ 4 C═S H Cl H Cl 4 C═S Cl H Cl H 4 C═S Cl H H Cl 4 C═SH Br H Br 4 C═S Br H Br H 4 C═S Br H H Br 5 C═S H I H I 5 C═S I H I H 5C═S I H H I 5 C═S H CH₃ H CH₃ 5 C═S CH H CH₃ H 5 C═S CH H H CH₃ 2 C═S HC₂H₅ H C₂H₅ 2 C═S C₂H₅ H C₂H₅ H 2 C═S C₂H₅ H H C₂H₅ 2 C═S H i-C₃H₇ Hi-C₃H₇ 2 C═S i-C₃H₇ H i-C₃H₇ H 2 C═S i-C₃H₇ H H i-C₃H₇ 3 CH₂ H Cl H Cl 3CH₂ Cl H Cl H 3 CH₂ Cl H H Cl 3 CH₂ H Br H Br 3 CH₂ Br H Br H 3 CH₂ Br HH Br 4 CH₂ H I H I 4 CH₂ I H I H 4 CH₂ I H H I 4 CH₂ H CH₃ H CH₃ 4 CH₂CH₃ H CH₃ H 4 CH₂ CH₃ H H CH₃ 5 CH₂ H C₂H₅ H C₂H₅ 5 CH₂ C₂H₅ H C₂H₅ H 5CH₂ C₂H₅ H H C₂H₅ 5 CH₂ H i-C₃H₇ H i-C₃H₇ 5 CH₂ i-C₃H₇ H i-C₃H₇ H 5 CH₂i-C₃H₇ H H i-C₃H₇ 2 CH—OH H Cl H Cl 2 CH—OH Cl H Cl H 2 CH—OH Cl H H Cl2 CH—OH H Br H Br 2 CH—OH Br H Br H 2 CH—OH Br H H Br 3 CH—OH H I H I 3CH—OH I H I H 3 CH—OH I H H I 3 CH—OH H CH₃ H CH₃ 3 CH—OH CH₃ H CH₃ H 3CH—OH CH₃ H H CH₃ 4 CH—OH H C₂H₅ H C₂H₅ 4 CH—OH C₂H₅ H C₂H₅ H 4 CH—OHC₂H₅ H H C₂H₅ 4 CH—OH H i-C₃H₇ H i-C₃H₇ 4 CH—OH i-C₃H₇ H i-C₃H₇ H 4CH—OH i-C₃H₇ H H i-C₃H₇ 5 CH—F H Cl H Cl 5 CH—F Cl H Cl H 5 CH—F Cl H HCl 5 CH—F H Br H Br 5 CH—F Br H Br H 5 CH—F Br H H Br 2 CH—F H I H I 2CH—F I H I H 2 CH—F I H H I 2 CH—F H CH₃ H CH₃ 2 CH—F CH₃ H CH₃ H 2 CH—FCH₃ H H CH₃ 3 CH—F H C₂H₅ H C₂H₅ 3 CH—F C₂H₅ H C₂H₅ H 3 CH—F C₂H₅ H HC₂H₅ 3 CH—F H i-C₃H₇ H i-C₃H₇ 3 CH—F i-C₃H₇ H i-C₃H₇ H 3 CH—F i-C₃H₇ H Hi-C₃H₇ 4 S H Cl Cl Cl 4 S Cl Cl H Cl 4 S H Br Br Br 4 S Br Br H Br 4 S HI I I 4 S I I H I 5 S H CH₃ CH₃ CH₃ 5 S CH CH₃ H CH₃ 5 S H C₂H₅ C₂H₅C₂H₅ 5 S C₂H₅ C₂H₅ H C₂H₅ 5 S H i-C₃H₇ i-C₃H₇ i-C₃H₇ 5 S i-C₃H₇ i-C₃H₇ Hi-C₃H₇ 2 O H Cl Cl Cl 2 O Cl Cl H Cl 2 O H Br Br Br 2 O Br Br H Br 2 O HI I I 2 O I I H I 3 O H CH₃ CH₃ CH₃ 3 O CH₃ CH₃ H CH₃ 3 O H C₂H₅ C₂H₅C₂H₅ 3 O C₂H₅ C₂H₅ H C₂H₅ 3 O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 O i-C₃H₇ i-C₃H₇ Hi-C₃H₇ 4 S═O H Cl Cl Cl 4 S═O Cl Cl H Cl 4 S═O H Br Br Br 4 S═O Br Br HBr 4 S═O H I I I 4 S═O I I H I 5 S═O H CH₃ CH₃ CH₃ 5 S═O CH CH₃ H CH₃ 5S═O H C₂H₅ C₂H₅ C₂H₅ 5 S═O C₂H₅ C₂H₅ H C₂H₅ 5 S═O H i-C₃H₇ i-C₃H₇ i-C₃H₇5 S═O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 O═S═O H Cl Cl Cl 2 O═S═O Cl Cl H Cl 2O═S═O H Br Br Br 2 O═S═O Br Br H Br 2 O═S═O H I I I 2 O═S═O I I H I 3O═S═O H CH₃ CH₃ CH₃ 3 O═S═O CH CH₃ H CH₃ 3 O═S═O H C₂H₅ C₂H₅ C₂H₅ 3O═S═O C₂H₅ C₂H₅ H C₂H₅ 3 O═S═O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 O═S═O i-C₃H₇i-C₃H₇ H i-C₃H₇ 4 NH H Cl Cl Cl 4 NH Cl Cl H Cl 4 NH H Br Br Br 4 NH BrBr H Br 4 NH H I I I 4 NH I I H I 5 NH H CH₃ CH₃ CH₃ 5 NH CH₃ CH₃ H CH₃5 NH H C₂H₅ C₂H₅ C₂H₅ 5 NH C₂H₅ C₂H₅ H C₂H₅ 5 NH H i-C₃H₇ i-C₃H₇ i-C₃H₇5 NH i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 C═O H Cl Cl Cl 2 C═O Cl Cl H Cl 2 C═O H BrBr Br 2 C═O Br Br H Br 2 C═O H I I I 2 C═O I I H I 3 C═O H CH₃ CH₃ CH₃ 3C═O CH₃ CH₃ H CH₃ 3 C═O H C₂H₅ C₂H₅ C₂H₅ 3 C═O C₂H₅ C₂H₅ H C₂H₅ 3 C═O Hi-C₃H₇ i-C₃H₇ i-C₃H₇ 3 C═O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 4 C═S H Cl Cl Cl 4 C═SCl Cl H Cl 4 C═S H Br Br Br 4 C═S Br Br H Br 4 C═S H I I I 4 C═S I I H I5 C═S H CH₃ CH₃ CH₃ 5 C═S CH₃ CH₃ H CH₃ 5 C═S H C₂H₅ C₂H₅ C₂H₅ 5 C═SC₂H₅ C₂H₅ H C₂H₅ 5 C═S H i-C₃H₇ i-C₃H₇ i-C₃H₇ 5 C═S i-C₃H₇ i-C₃H₇ Hi-C₃H₇ 2 CH₂ H Cl Cl Cl 2 CH₂ Cl Cl H Cl 2 CH₂ H Br Br Br 2 CH₂ Br Br HBr 2 CH₂ H I I I 2 CH₂ I I H I 3 CH₂ H CH₃ CH₃ CH₃ 3 CH₂ CH₃ CH₃ H CH₃ 3CH₂ H C₂H₅ C₂H₅ C₂H₅ 3 CH₂ C₂H₅ C₂H₅ H C₂H₅ 3 CH₂ H i-C₃H₇ i-C₃H₇ i-C₃H₇3 CH₂ i-C₃H₇ i-C₃H₇ H i-C₃H₇ 4 CH—OH H Cl Cl Cl 4 CH—OH Cl Cl H Cl 4CH—OH H Br Br Br 4 CH—OH Br Br H Br 4 CH—OH H I I I 4 CH—OH I I H I 5CH—OH H CH₃ CH₃ CH₃ 5 CH—OH CH₃ CH₃ H CH₃ 5 CH—OH H C₂H₅ C₂H₅ C₂H₅ 5CH—OH C₂H₅ C₂H₅ H C₂H₅ 5 CH—OH H i-C₃H₇ i-C₃H₇ i-C₃H₇ 5 CH—OH i-C₃H₇i-C₃H₇ H i-C₃H₇ 2 CH—F H Cl Cl Cl 2 CH—F Cl Cl H Cl 2 CH—F H Br Br Br 2CH—F Br Br H Br 2 CH—F H I I I 2 CH—F I I H I 3 CH—F H CH₃ CH₃ CH₃ 3CH—F CH₃ CH₃ H CH₃ 3 CH—F H C₂H₅ C₂H₅ C₂H₅ 3 CH—F C₂H₅ C₂H₅ H C₂H₅ 3CH—F H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH—F i-C₃H₇ i-C₃H₇ H i-C₃H₇ 4 S I H Cl H 4S═O I H H Cl 4 O═S═O Br H Cl H 4 CH₂ Br H H Cl 4 C═O Br H I H 4 C═S Br HH I 5 CH—OH I H Br H 5 CH—F I H H Br 5 O I Cl H Cl 5 NH Br Cl H Cl

5.3.2 Grp94 Inhibitors of Formula (II)

In one aspect, the disclosure encompasses purine-scaffold compounds thatare substituted at the 8-position with a linker group bonded to a2,4,6-tri-substituted aryl group and are further substituted at the N-9position. Such compounds are represented schematically in Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

(a) Y is —C(R^(Y))₂—, —S—, —NR—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—;

(c) Z² is —N— or —CR¹⁰—, wherein R¹⁰ is H or unsubstituted orsubstituted —(C₁-C₆)aliphatic;

(d) X¹ is —H, -halo, —N(R)₂, —OR, —CN, or unsubstituted or substituted—(C₁-C₆)aliphatic;

(e) each of X², X⁴, and X⁶ are independently —H, -halo, —SR, —N(R)₂,—OR, —CN, —NO₂, —CN, —C(O)R, —C(O)₂R, —S(O)R, —S(O)₂R, —C(O)N(R)₂,—SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)SO₂R, —OC(O)N(R)₂, unsubstituted orsubstituted —(C₁-C₆)aliphatic, or an unsubstituted or substituted groupselected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and(5- or 6-membered)heterocyclic aromatic or heterocyclic non-aromaticgroup;

(f) R¹ is —(C₁-C₆)aliphatic-N⁺—(R²)(R³)(R⁴), —(C₁-C₆)aliphatic-N—R³R⁴,—(C₁-C₆)aliphatic-C(═O)N—R³R⁴, —(C₁-C₆)aliphatic-R³R⁴,—(C₁-C₆)aliphatic-R²R³R⁴, —(C₁-C₆)aliphatic-N—CR²R³R⁴,—(C₁-C₆)aliphatic-C(halo)₃, —(C₁-C₆)aliphatic-alkenyl,—(C₁-C₆)aliphatic-alkynyl, —(C₁-C₆)aliphatic-(C₃-C₈)cycloalkyl,—(C₁-C₆)aliphatic-(C₃-C₈)heterocycloalkyl, —(C₁-C₆)aliphatic-phenyl,—(C₁-C₆)aliphatic-(5 or 6-membered)heteroaryl, —(C₁-C₆)aliphatic-cyano,with the proviso that when all of R²-R⁴ are present the compound furthercomprises a pharmaceutically acceptable counter ion;

(g) R² and R³ are independently hydrogen, —N(R)₂, —CH₂CH(OH)R⁴,—CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴, or unsubstituted or substituted—(C₁-C₆)aliphatic, or R³ and R⁴ form an unsubstituted or substituted 3-to 7-membered heterocyclic ring when taken together with the nitrogen towhich they are attached;

(h) each R^(Y) is independently R, —OR, or halo;

(i) R⁴ is hydrogen, halogen, or unsubstituted or substituted—(C₁-C₆)aliphatic; and

(j) each R is independently hydrogen, unsubstituted C₁₋₆ aliphatic, orC₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂;

wherein each substituted group is substituted with one or more groupsselected from halo, —N(R)₂, —OR, —CN, oxo, unsubstituted C₁₋₆ aliphatic,or C₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂.

In some embodiments, a compound of formula (II) or pharmaceuticallyacceptable salt thereof is defined wherein:

(a) Y is —CH₂—, —S—, —NH—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—;

(c) Z² is —CH—, —N—, or —CR¹⁰—, wherein R¹⁰ is —(C₁-C₆)alkyl;

(d) X¹ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, or—OCH₂(halo);

(e) each of X², X⁴ and X⁶ are independently —H, -halo, —NH₂, —CN,—(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, —OCH₂(halo), or a (5- or6-membered)aryl, heterocyclic aromatic, or non-aromatic group selectedfrom pyridyl, furyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl, phenyl,benzyl, thiazolidinyl, thiadiazolyl, thiazolyl, isoxazolyl, pyrazolyl,isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl,2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl;

(f) R¹ is —(CH₂)_(m)—N⁺—(R²)(R³)(R⁴), —(CH₂)_(m)—N—R³R⁴,—(CH₂)_(m)—C(═O)N—R³R⁴, —(CH₂)_(m)—R³R⁴, —(CH₂)_(m)—C(halo)₃,—(CH₂)_(m)-alkenyl, —(CH₂)_(m)-alkenyl-CH₃, —(CH₂)_(m)-alkynyl,—(CH₂)_(m)-alkynyl-CH₃, —(CH₂)_(m)—(C₃-C₈)cycloalkyl,—(CH₂)_(m)—(C₃-C₈)heterocycloalkyl, —(CH₂)_(m)-phenyl, —(CH₂)_(m)-(5 or6-membered)heteroaryl, —(CH₂)_(m)-cyano, where m is 1, 2, 3, 4 or 5 andwhere the cyloalkyl, heterocycle or phenyl is unsubstituted orsubstituted with one or more X¹ groups, with the proviso that when allof R²-R⁴ are present the compound further comprises a pharmaceuticallyacceptable counter ion;

(g) R² and R³ are independently hydrogen, methyl, ethyl, ethenyl,ethynyl, propyl, butyl, pentyl, hexyl, isopropyl, t-butyl, isobutyl,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —CH₂C(halo)₃, —CHCH(halo)₂,CHCH₂(halo), —CH₂OH, —CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH₂CH(CH₃)OH,—C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH, —CH(CH₃)CH(OH)R⁴, —CH₂CH(OH)R⁴,—CH₂SO₂NHR⁴, —CH₂SO₂NHR⁴, or R² and R³ form an unsubstituted orsubstituted aziridine, azetidine, pyrrolidine, piperazine, or piperidinering when taken together with the nitrogen to which they are attached;and

(h) R⁴ is hydrogen, methyl, ethyl, isopropyl, t-butyl, isobutyl, or—C(halo)₃.

In one embodiment, Z, Z² and Z³ are —N—. In another embodiment, Z¹ andZ³ are —N— and Z² is —CH—. In another embodiment, Z¹ is —CH— and Z² andZ³ are —N—.

In another embodiment, Y is —S—, —CH₂—, or

In another embodiment, Y is S or

In another embodiment, Y is —S— or —CH₂—. In another embodiment, Y is—S— or —O—. In another embodiment, Y is —S—. In another embodiment, Y is—CH₂—. In another embodiment, Y is

In some embodiments, Y is —C(R^(Y))₂—, wherein each R^(Y) isindependently hydrogen, —OH, or halo.

In certain embodiment, R¹ is —(CH₂)_(m)—N—(R³)(R⁴). In one suchembodiment, R¹ is —(CH₂)₂—N—(R³)(R⁴). In another such embodiment, R¹ is—(CH₂)₃—N—(R³)(R⁴). In another such embodiment, R¹ is—(CH₂)₂—N—(R³)(R⁴), R³ is H and R⁴ is isopropyl or isobutyl. In anothersuch embodiment, R¹ is —(CH₂)₂—N—(R³)(R⁴), R³ is —H and R⁴ is isopropyl.In another such embodiment, R¹ is —(CH₂)₃—N—(R³)(R⁴), R³ is —H and R⁴ isisobutyl. In another such embodiment, R¹ is —(CH₂)₃—N—(R³)(R⁴), R³ is —Hand R⁴ is isopropyl. It will be understood, that in these embodiments,the amine functionality may exist as a free base or as an acid additionsalt. Acid addition salts can be prepared by addition of a suitableacid, as is well understood in the art. In particular embodiments, theacid addition salt may be a hydrochloride salt, a phosphate salt, asulfate salt, a lactate salt, a citrate salt, a succinate salt, amesylate salt, a tartrate salt, a lactobionate salt, a benzene sulfonicacid salt, a para-toluenesulfonic acid salt, or a fumaric acid-salt. Inanother embodiment, the acid addition salt is a hydrochloride salt or asulfate salt. In another embodiment, the acid addition salt is ahydrochloride salt. In another embodiment, the acid addition salt is asulfate salt. In another embodiment, the acid addition salt is aphosphate salt.

In certain embodiments, R¹ is —(CH₂)_(m)—CF₃. In one such embodiment, R¹is —(CH₂)₃—CF₃. In another such embodiment, R¹ is —(CH₂)₄—CF₃.

In some embodiments, R¹ is is —(CH₂)₃—CCH.

In some embodiments, R² or R³ is —CH₂NHSO₂R⁴.

In some embodiments, R² and R³ are independently hydrogen, methyl,ethyl, ethenyl, ethynyl, propyl, butyl, pentyl, hexyl, isopropyl,t-butyl, isobutyl, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —CH₂C(halo)₃,—CHCH(halo)₂, CHCH₂(halo), —CH₂OH, —CH₂CH₂OH, —CH₂C(CH₃)₂OH,—CH₂CH(CH₃)OH, —C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH, —CH(CH₃)CH(OH)R⁴,—CH₂CH(OH)R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴ or R² and R³ form anunsubstituted or substituted aziridine, azetidine, pyrrolidine,piperazine, or piperidine ring when taken together with the nitrogen towhich they are attached.

In other embodiments, the Grp94 inhibitors of Formula (II) have one ofthe Formula of Table 4, wherein each substituent is as defined above anddescribed in classes and subclasses herein, both singly and incombination.

TABLE 4 Formula Compound IIA

IIB

IIC

IID

IIE

IIF

IIG

IIH

III

IIJ

IIK

IIL

IIM

IIN

IIO

IIP

IIQ

IIR

IIS

IIT

Illustrative compounds of Formula (II) are listed below in Table 5.

TABLE 5

and pharmaceutically acceptable salts thereof, where: m R⁴ Y X² X⁴ X⁶ 2CH(CH₃)₂ S Cl Cl Cl 2 CH₂CH₂OH S CH₃ CH₃ CH₃ 2 CH₂CH(CH₃)OH S C₂H₅ C₂H₅C₂H₅ 2 CH(CH₃)CH₂OH S i-C₃H₇ i-C₃H₇ i-C₃H₇ 2 CH(CH₃)CH(CH₃)OH S CH₃ ClCl 2 C(CH₃)₂CH₂OH S CH₃ CH₃ Cl 2 CH₂C(CH₃)₂OH S CH₃ Cl CH₃ 2 CH₂CHF₂ SC₂H₅ Cl Cl 2 CH₂CF₃ S C₂H₅ C₂H₅ Cl 3 CH₂CH(CH₃)₂ S C₂H₅ Cl C₂H₅ 3CH₂C(CH₃)₃ S CH₃ Br Br 3 H₂C—C≡N S CH₃ CH₃ Br 3 H₂C—C≡CH S CH₃ Br CH₃ 3CH(CH₃)₂ S C₂H₅ I I 3 CH₂CH₂OH S C₂H₅ C₂H₅ I 3 CH₂CH(CH₃)OH S C₂H₅ IC₂H₅ 3 CH(CH₃)CH₂OH S C₂H₅ Br Cl 3 CH(CH₃)CH(CH₃)OH S C₂H₅ C₂H₅ Br 3C(CH₃)₂CH₂OH S C₂H₅ Br C₂H₅ 2 CH₂C(CH₃)₂OH S═O Cl Cl Cl 2 CH₂CHF₂ S═OCH₃ CH₃ CH₃ 2 CH₂CF₃ S═O C₂H₅ C₂H₅ C₂H₅ 2 CH₂CH(CH₃)₂ S═O i-C₃H₇ i-C₃H₇i-C₃H₇ 2 CH₂C(CH₃)₃ S═O CH₃ Cl Cl 2 H₂C—C≡N S═O CH₃ CH₃ Cl 2 H₂C—C≡CHS═O CH₃ Cl CH₃ 2 CH(CH₃)₂ S═O C₂H₅ Cl Cl 2 CH₂CH₂OH S═O C₂H₅ C₂H₅ Cl 3CH₂CH(CH₃)OH S═O C₂H₅ Cl C₂H₅ 3 CH(CH₃)CH₂OH S═O CH₃ Br Br 3CH(CH₃)CH(CH₃)OH S═O CH₃ CH₃ Br 3 C(CH₃)₂CH₂OH S═O CH₃ Br CH₃ 3CH₂C(CH₃)₂OH S═O C₂H₅ I I 3 CH₂CHF₂ S═O C₂H₅ C₂H₅ I 3 CH₂CF₃ S═O C₂H₅ IC₂H₅ 3 CH₂CH(CH₃)₂ S═O C₂H₅ Br Cl 3 CH₂C(CH₃)₃ S═O C₂H₅ C₂H₅ Br 3H₂C—C≡N S═O C₂H₅ Br C₂H₅ 3 H₂C—C≡CH O═S═O Cl Cl Cl 2 CH(CH₃)₂ O═S═O CH₃CH₃ CH₃ 2 CH₂CH₂OH O═S═O C₂H₅ C₂H₅ C₂H₅ 2 CH₂CH(CH₃)OH O═S═O i-C₃H₇i-C₃H₇ i-C₃H₇ 2 CH(CH₃)CH₂OH O═S═O CH₃ Cl Cl 2 CH(CH₃)CH(CH₃)OH O═S═OCH₃ CH₃ Cl 2 C(CH₃)₂CH₂OH O═S═O CH₃ Cl CH₃ 2 CH₂C(CH₃)₂OH O═S═O C₂H₅ ClCl 2 CH₂CHF₂ O═S═O C₂H₅ C₂H₅ Cl 2 CH₂CF₃ O═S═O C₂H₅ Cl C₂H₅ 3CH₂CH(CH₃)₂ O═S═O CH₃ Br Br 3 CH₂C(CH₃)₃ O═S═O CH₃ CH₃ Br 3 H₂C—C≡NO═S═O CH₃ Br CH₃ 3 H₂C—C≡CH O═S═O C₂H₅ I I 3 CH(CH₃)₂ O═S═O C₂H₅ C₂H₅ I3 CH₂CH₂OH O═S═O C₂H₅ I C₂H₅ 3 CH₂CH(CH₃)OH O═S═O C₂H₅ Br Cl 3CH(CH₃)CH₂OH O═S═O C₂H₅ C₂H₅ Br 3 CH(CH₃)CH(CH₃)OH O═S═O C₂H₅ Br C₂H₅ 2C(CH₃)₂CH₂OH NH Cl Cl Cl 2 CH₂C(CH₃)₂OH NH CH₃ CH₃ CH₃ 2 CH₂CHF₂ NH C₂H₅C₂H₅ C₂H₅ 2 CH₂CF₃ NH i-C₃H₇ i-C₃H₇ i-C₃H₇ 2 CH₂CH(CH₃)₂ NH CH₃ Cl Cl 2CH₂C(CH₃)₃ NH CH₃ CH₃ Cl 2 H₂C—C≡N NH CH₃ Cl CH₃ 2 H₂C—C≡CH NH C₂H₅ ClCl 2 CH(CH₃)₂ NH C₂H₅ C₂H₅ Cl 3 CH₂CH₂OH NH C₂H₅ Cl C₂H₅ 3 CH₂CH(CH₃)OHNH CH₃ Br Br 3 CH(CH₃)CH₂OH NH CH₃ CH₃ Br 3 CH(CH₃)₂ NH CH₃ Br CH₃ 3CH₂CH₂OH NH C₂H₅ I I 3 CH₂CH(CH₃)OH NH C₂H₅ C₂H₅ I 3 CH(CH₃)CH₂OH NHC₂H₅ I C₂H₅ 3 CH(CH₃)CH(CH₃)OH NH C₂H₅ Br Cl 3 C(CH₃)₂CH₂OH NH C₂H₅ C₂H₅Br 3 CH₂C(CH₃)₂OH NH C₂H₅ Br C₂H₅ 3 CH₂CHF₂ O Cl Cl Cl 2 CH₂CF₃ O CH₃CH₃ CH₃ 2 CH₂CH(CH₃)₂ O C₂H₅ C₂H₅ C₂H₅ 2 CH₂C(CH₃)₃ O i-C₃H₇ i-C₃H₇i-C₃H₇ 2 H₂C—C≡N O CH₃ Cl Cl 2 H₂C—C≡CH O CH₃ CH₃ Cl 2 CH(CH₃)₂ O CH₃ ClCH₃ 2 CH₂CH₂OH O C₂H₅ Cl Cl 2 CH₂CH(CH₃)OH O C₂H₅ C₂H₅ Cl 2 CH(CH₃)CH₂OHO C₂H₅ Cl C₂H₅ 3 CH(CH₃)CH(CH₃)OH O CH₃ Br Br 3 C(CH₃)₂CH₂OH O CH₃ CH₃Br 3 CH₂C(CH₃)₂OH O CH₃ Br CH₃ 3 CH₂CHF₂ O C₂H₅ I I 3 CH₂CF₃ O C₂H₅ C₂H₅I 3 CH₂CH(CH₃)₂ O C₂H₅ I C₂H₅ 3 CH₂C(CH₃)₃ O C₂H₅ Br Cl 3 H₂C—C≡N O C₂H₅C₂H₅ Br 3 H₂C—C≡CH O C₂H₅ Br C₂H₅ 2 CH(CH₃)₂ CH₂ Cl Cl Cl 2 CH₂CH₂OH CH₂CH₃ CH₃ CH₃ 2 CH₂CH(CH₃)OH CH₂ C₂H₅ C₂H₅ C₂H₅ 2 CH(CH₃)CH₂OH CH₂ i-C₃H₇i-C₃H₇ i-C₃H₇ 2 CH(CH₃)CH(CH₃)OH CH₂ CH₃ Cl Cl 2 C(CH₃)₂CH₂OH CH₂ CH₃CH₃ Cl 2 CH₂C(CH₃)₂OH CH₂ CH₃ Cl CH₃ 2 CH₂CHF₂ CH₂ C₂H₅ Cl Cl 2 CH₂CF₃CH₂ C₂H₅ C₂H₅ Cl 3 CH₂CH(CH₃)₂ CH₂ C₂H₅ Cl C₂H₅ 3 CH₂C(CH₃)₃ CH₂ CH₃ BrBr 3 H₂C—C≡N CH₂ CH₃ CH₃ Br 3 H₂C—C≡CH CH₂ CH₃ Br CH₃ 3 CH(CH₃)₂ CH₂C₂H₅ I I 3 CH₂CH₂OH CH₂ C₂H₅ C₂H₅ I 3 CH₂CH(CH₃)OH CH₂ C₂H₅ I C₂H₅ 3CH(CH₃)CH₂OH CH₂ C₂H₅ Br Cl 3 CH(CH₃)CH(CH₃)OH CH₂ C₂H₅ C₂H₅ Br 3C(CH₃)₂CH₂OH CH₂ C₂H₅ Br C₂H₅ 2 CH₂C(CH₃)₂OH C═O Cl Cl Cl 2 CH₂CHF₂ C═OCH₃ CH₃ CH₃ 2 CH₂CF₃ C═O C₂H₅ C₂H₅ C₂H₅ 2 CH₂CH(CH₃)₂ C═O i-C₃H₇ i-C₃H₇i-C₃H₇ 2 CH₂C(CH₃)₃ C═O CH₃ Cl Cl 2 H₂C—C≡N C═O CH₃ CH₃ Cl 2 H₂C—C≡CHC═O CH₃ Cl CH₃ 2 CH(CH₃)₂ C═O C₂H₅ Cl Cl 2 CH₂CH₂OH C═O C₂H₅ C₂H₅ Cl 3CH₂CH(CH₃)OH C═O C₂H₅ Cl C₂H₅ 3 CH(CH₃)CH₂OH C═O CH₃ Br Br 3CH(CH₃)CH(CH₃)OH C═O CH₃ CH₃ Br 3 C(CH₃)₂CH₂OH C═O CH₃ Br CH₃ 3CH₂C(CH₃)₂OH C═O C₂H₅ I I 3 CH₂CHF₂ C═O C₂H₅ C₂H₅ I 3 CH₂CF₃ C═O C₂H₅ IC₂H₅ 3 CH₂CH(CH₃)₂ C═O C₂H₅ Br Cl 3 CH₂C(CH₃)₃ C═O C₂H₅ C₂H₅ Br 3H₂C—C≡N C═O C₂H₅ Br C₂H₅ 3 H₂C—C≡CH C═S Cl Cl Cl 2 CH(CH₃)₂ C═S CH₃ CH₃CH₃ 2 CH₂CH₂OH C═S C₂H₅ C₂H₅ C₂H₅ 2 CH₂CH(CH₃)OH C═S i-C₃H₇ i-C₃H₇i-C₃H₇ 2 CH(CH₃)CH₂OH C═S CH₃ Cl Cl 2 CH(CH₃)CH(CH₃)OH C═S CH₃ CH₃ Cl 2C(CH₃)₂CH₂OH C═S CH₃ Cl CH₃ 2 CH₂C(CH₃)₂OH C═S C₂H₅ Cl Cl 2 CH₂CHF₂ C═SC₂H₅ C₂H₅ Cl 2 CH₂CF₃ C═S C₂H₅ Cl C₂H₅ 3 CH₂CH(CH₃)₂ C═S CH₃ Br Br 3CH₂C(CH₃)₃ C═S CH₃ CH₃ Br 3 H₂C—C≡N C═S CH₃ Br CH₃ 3 H₂C—C≡CH C═S C₂H₅ II 3 CH(CH₃)₂ C═S C₂H₅ C₂H₅ I 3 CH₂CH₂OH C═S C₂H₅ I C₂H₅ 3 CH₂CH(CH₃)OHC═S C₂H₅ Br Cl 3 CH(CH₃)CH₂OH C═S C₂H₅ C₂H₅ Br 3 CH(CH₃)CH(CH₃)OH C═SC₂H₅ Br C₂H₅ 2 C(CH₃)₂CH₂OH CH—OH Cl Cl Cl 2 CH₂C(CH₃)₂OH CH—OH CH₃ CH₃CH₃ 2 CH₂CHF₂ CH—OH C₂H₅ C₂H₅ C₂H₅ 2 CH₂CF₃ CH—OH i-C₃H₇ i-C₃H₇ i-C₃H₇ 2CH₂CH(CH₃)₂ CH—OH CH₃ Cl Cl 2 CH₂C(CH₃)₃ CH—OH CH₃ CH₃ Cl 2 H₂C—C≡NCH—OH CH₃ Cl CH₃ 2 H₂C—C≡CH CH—OH C₂H₅ Cl Cl 2 CH(CH₃)₂ CH—OH C₂H₅ C₂H₅Cl 3 CH₂CH₂OH CH—OH C₂H₅ Cl C₂H₅ 3 CH₂CH(CH₃)OH CH—OH CH₃ Br Br 3CH(CH₃)CH₂OH CH—OH CH₃ CH₃ Br 3 CH(CH₃)CH(CH₃)OH CH—OH CH₃ Br CH₃ 3C(CH3)₂CH₂OH CH—OH C₂H₅ I I 3 CH₂C(CH₃)₂OH CH—OH C₂H₅ C₂H₅ I 3 CH₂CHF₂CH—OH C₂H₅ I C₂H₅ 3 CH₂CF₃ CH—OH C₂H₅ Br Cl 3 CH₂CH(CH₃)₂ CH—OH C₂H₅C₂H₅ Br 3 CH₂C(CH₃)₃ CH—OH C₂H₅ Br C₂H₅ 2 H₂C—C≡N CH—F Cl Cl Cl 2H₂C—C≡CH CH—F CH₃ CH₃ CH₃ 2 CH(CH₃)₂ CH—F C₂H₅ C₂H₅ C₂H₅ 2 CH₂CH₂OH CH—Fi-C₃H₇ i-C₃H₇ i-C₃H₇ 2 CH₂CH(CH₃)OH CH—F CH₃ Cl Cl 2 CH(CH₃)CH₂OH CH—FCH₃ CH₃ Cl 2 CH(CH₃)CH(CH₃)OH CH—F CH₃ Cl CH₃ 2 C(CH₃)₂CH₂OH CH—F C₂H₅Cl Cl 2 CH₂C(CH₃)₂OH CH—F C₂H₅ C₂H₅ Cl 3 CH₂CHF₂ CH—F C₂H₅ Cl C₂H₅ 3CH₂CF₃ CH—F CH₃ Br Br 3 CH₂CH(CH₃)₂ CH—F CH₃ CH₃ Br 3 CH₂C(CH₃)₃ CH—FCH₃ Br CH₃ 3 H₂C—C≡N CH—F C₂H₅ I I 3 H₂C—C≡CH CH—F C₂H₅ C₂H₅ I 3CH(CH₃)₂ CH—F C₂H₅ I C₂H₅ 3 CH₂CH₂OH CH—F C₂H₅ Br Cl 3 CH₂CH(CH₃)OH CH—FC₂H₅ C₂H₅ Br 3 CH(CH₃)CH₂OH CH—F C₂H₅ Br C₂H₅

5.3.3 Grp94 Inhibitors of Formula (III)

In one aspect, the disclosure encompasses purine-scaffold compounds thatare substituted at the 8-position with a linker group bonded to abicyclic group and are further substituted at the N-9 position. Suchcompounds are represented schematically in Formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

(a) Y is —C(R^(Y))₂-, —S—, —NR—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—;

(c) Z² is —N— or —CR¹⁰—, wherein R¹⁰ is H or unsubstituted orsubstituted —(C₁-C₆)aliphatic;

(d) each of Z⁶, Z⁷ and Z⁸ are independently —C— or —N—, with the provisothat at least one of Z⁶—Z⁸ is —C—;

(e) X¹ is —H, -halo, —N(R)₂, —OR, —CN, or unsubstituted or substituted—(C₁-C₆)aliphatic;

(f) each of X⁴, X⁵, and X⁶ are independently —H, -halo, —SR, —N(R)₂,—OR, —CN, —NO₂, —CN, —C(O)R, —C(O)₂R, —S(O)R, —S(O)₂R, —C(O)N(R)₂,—SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)SO₂R, —OC(O)N(R)₂, unsubstituted orsubstituted —(C₁-C₆)aliphatic, or an unsubstituted or substituted groupselected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and(5- or 6-membered)heterocyclic aromatic or heterocyclic non-aromaticgroup; with the provisos that X⁴ is absent when Z⁶ is a nitrogen, X⁵ isabsent when Z⁷ is a nitrogen and X⁶ is absent when Z⁸ is a nitrogen;

(g) R⁷ is —(C₁-C₆)aliphatic-N⁺—(R²)(R³)(R⁴), —(C₁-C₆)aliphatic-N—R³R⁴,—(C₁-C₆)aliphatic-C(═O)N—R³R⁴, —(C₁-C₆)aliphatic-R³R⁴,—(C₁-C₆)aliphatic-R²R³R⁴, —(C₁-C₆)aliphatic-N—CR²R³R⁴,—(C₁-C₆)aliphatic-C(halo)₃, —(C₁-C₆)aliphatic-alkenyl,—(C₁-C₆)aliphatic-alkynyl, —(C₁-C₆)aliphatic-(C₃-C₈)cycloalkyl,—(C₁-C₆)aliphatic-(C₃-C₈)heterocycloalkyl, —(C₁-C₆)aliphatic-phenyl,—(C₁-C₆)aliphatic-(5 or 6-membered)heteroaryl, —(C₁-C₆)aliphatic-cyano,with the proviso that when all of R²-R⁴ are present the compound furthercomprises a pharmaceutically acceptable counter ion;

(h) Q is fused benzo, fused (5- or 6-membered)heteroaryl, a fused 4 to7-membered cyloalkyl ring or a fused 4- to 7-membered non-aromaticheterocyclic ring;

(i) R² and R³ are independently hydrogen, —N(R)₂, —CH₂CH(OH)R⁴,—CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴, or unsubstituted or substituted—(C₁-C₆)aliphatic, or R³ and R⁴ form an unsubstituted or substituted 3-to 7-membered heterocyclic ring when taken together with the nitrogen towhich they are attached;

(j) R⁴ is hydrogen, halogen, or unsubstituted or substituted—(C₁-C₆)aliphatic;

(k) each R⁸ is independently —H, -halo, —N(R)₂, —OR, —CN, or aunsubstituted or substituted selected from —CH₂-phenyl or—(C₁-C₆)aliphatic;

(l) each R^(Y) is independently R, —OR, or halo;

(m) a is an integer selected from 0, 1 and 2; and

(n) each R is independently hydrogen, unsubstituted C₁₋₆ aliphatic, orC₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂;

wherein each substituted group is substituted with one or more groupsselected from halo, —N(R)₂, —OR, —CN, oxo, unsubstituted C₁₋₆ aliphatic,or C₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂.

In some embodiments, a compound of formula (III) or pharmaceuticallyacceptable salt thereof is defined wherein:

(a) Y is —CH₂—, —S—, —N—, —O—,

(b) each of Z¹ and Z³ are independently —C— or —N—;

(c) Z² is —CH—, —N—, or —CR¹⁰—, wherein R¹⁰ is —(C₁-C₆)alkyl;

(d) each of Z⁶, Z⁷ and Z⁸ are independently —C— or —N—, with the provisothat at least one of Z⁶—Z⁸ is —C—;

(e) X¹ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, or—OCH₂(halo);

(f) each of X⁴, X⁵, and X⁶ are independently —H, -halo, —NH₂, —CN,—(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, —OCH₂(halo), or a (5- or6-membered)aryl, heterocyclic aromatic, or non-aromatic group selectedfrom pyridyl, furyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl,thiazolidinyl, thiadiazolyl, thiazolyl, isoxazolyl, pyrazolyl,isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl,2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl,with the provisos that X⁴ is absent when Z⁶ is a nitrogen, X⁵ is absentwhen Z⁷ is a nitrogen and X⁶ is absent when Z⁸ is a nitrogen;

(g) R⁷ is —(CH₂)_(m)—N⁺—(R²)(R³)(R⁴), —(CH₂)_(m)—N—R³R⁴,—(CH₂)_(m)—C(═O)N—R³R⁴, —(CH₂)_(m)R³R⁴, —(CH₂)_(m)—C(halo)₃,—(CH₂)_(m)-alkenyl, (CH₂)_(m)-alkenyl-CH₃, —(CH₂)_(m)-alkynyl,(CH₂)_(m)-alkynyl-CH₃, (CH₂)_(m)—(C₃-C₈)cycloalkyl,—(CH₂)_(m)—(C₃-C₈)heterocycloalkyl, —(CH₂)_(m)-phenyl, —(CH₂)_(m)-(5 or6-membered)heteroaryl, —(CH₂)_(m)-cyano, where m is 1, 2, 3, 4 or 5 andwhere the cyloalkyl, heterocycle or phenyl is unsubstituted orsubstituted with one or more X¹ groups, with the proviso that when allof R²-R⁴ are present the compound further comprises a pharmaceuticallyacceptable counter ion;

(h) Q is fused benzo, fused (5- or 6-membered)heteroaryl, a fused 4 to7-membered cyloalkyl ring or a fused 4- to 7-membered non-aromaticheterocyclic ring selected from pyrrolo, pyridino, pyrimidino, pyrazino,pyridazino, oxadiazolo, thiadiazolo, dioxolano, imidazolo, orimidazo[1,2-a]pyridine;

(i) R² and R³ are independently hydrogen, methyl, ethyl, ethenyl,ethynyl, propyl, butyl, pentyl, hexyl, isopropyl, t-butyl, isobutyl,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —CH₂C(halo)₃, —CHCH(halo)₂,CHCH₂(halo), —CH₂OH, —CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH₂CH(CH₃)OH,—C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH, —CH(CH₃)CH(OH)R⁴, —CH₂CH(OH)R⁴,—CH₂SO₂NHR⁴, —CH₃SO₂NHR⁴ or R² and R³ form an unsubstituted orsubstituted aziridine, azetidine, pyrrolidine, piperazine, or piperidinering when taken together with the nitrogen to which they are attached;

(j) R⁴ is hydrogen, methyl, ethyl, isopropyl, t-butyl, isobutyl, or—C(halo)₃;

(k) R⁸ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, and—OCH₂(halo); and

(l) a is an integer selected from 0, 1 and 2.

In one embodiment, Z¹, Z² and Z³ are —N—. In another embodiment, Z¹ andZ³ are —N— and Z² is —CH—. In another embodiment, Z¹ is —C— and Z² andZ³ are —N—.

In another embodiment, Z⁶, Z⁷ and Z⁸ are —C—. In another embodiment, Z⁶is —N— and Z⁷ and Z⁸ are —C—.

In another embodiment, Y is —S—, —CH₂—, or

In another embodiment, Y is S or

In another embodiment, Y is —S— or —CH₂—. In another embodiment, Y is—S— or —O—. In another embodiment, Y is —S—. In another embodiment, Y is—CH₂—. In another embodiment, Y is

In some embodiments, Y is —C(R^(Y))₂-, wherein each R^(Y) isindependently hydrogen, —OH, or halo.

In some embodiments, R² or R³ is —CH₂NHSO₂R⁴.

In some embodiments, R² and R³ are independently hydrogen, —N(R)₂,—CH₂CH(OH)R⁴, —CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴, or unsubstitutedor substituted —(C₁-C₆)aliphatic, or R³ and R⁴ form an unsubstituted orsubstituted 3- to 7-membered heterocyclic ring when taken together withthe nitrogen to which they are attached.

In some embodiments, R² and R³ are independently hydrogen, methyl,ethyl, ethenyl, ethynyl, propyl, butyl, pentyl, hexyl, isopropyl,t-butyl, isobutyl, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —CH₂C(halo)₃,—CHCH(halo)₂, CHCH₂(halo), —CH₂OH, —CH₂CH₂OH, —CH₂C(CH₃)₂OH,—CH₂CH(CH₃)OH, —C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH, —CH(CH₃)CH(OH)R⁴,—CH₂CH(OH)R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴ or R² and R³ form anunsubstituted or substituted aziridine, azetidine, pyrrolidine,piperazine, or piperidine ring when taken together with the nitrogen towhich they are attached.

In certain embodiments, R⁷ is —(CH₂)_(m)—N—(R³)(R⁴). In one suchembodiment, R⁷ is —(CH₂)₂—N—(R³)(R⁴). In another such embodiment, R⁷ is—(CH₂)₃—N—(R³)(R⁴). In another such embodiment, R⁷ is—(CH₂)₂—N—(R³)(R⁴), R³ is H and R⁴ is isopropyl or isobutyl. In anothersuch embodiment, R⁷ is —(CH₂)₃—N—(R³)(R⁴), R³ is H and R⁴ is isopropyl.In another such embodiment, R⁷ is —(CH₂)₃—N—(R³)(R⁴), R³ is H and R⁴ isisobutyl.

In certain embodiments, R⁷ is —(CH₂)_(m)—CF₃. In one such embodiment, R⁷is —(CH₂)₃—CF₃. In another such embodiment, R⁷ is —(CH₂)₄—CF₃.

In certain embodiments, R⁷ is —(CH₂)_(m)-alkenyl. In one suchembodiment, R⁷ is —(CH₂)₃-alkenyl. In another such embodiment, R⁷ is—(CH₂)₄-alkenyl.

In another embodiment, R⁷ is —(CH₂)₃-alkynyl. In another embodiment, R⁷is —(CH₂)₃—CCH. In another embodiment, R⁷ is —(CH₂)₄-alkynyl. In anotherembodiment, R⁷ is —(CH₂)_(m)-cyano In another embodiment, R⁷ is—(CH₂)₃-cyano. In another embodiment, R⁷ is —(CH₂)₄-cyano.

In another embodiment Q is benzo, pyrrolo, pyridino, pyrimidino,pyrazino, or pyridazino. In another embodiment, Q is benzo or pyridino,wherein preferably the 2- and 3-positions of the pyridino are fused tothe 6-membered, nitrogen-containing ring. In another embodiment, Q isbenzo. In another embodiment, Q is oxadiazolo, thiadiazolo, dioxolano orimidazolo. In another embodiment, Q is fused with an aryl ring to forman imidazo[1,2-a]pyridine ring.

In another embodiment Q is piperazinyl, piperidinyl, 2H-pyranyl,pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl,oxoimidazolidinyl, 2-oxopyrrolidinyl, thiomorpholinyl, or thiazolidinyl.

In another embodiment, Q is cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl.

In other embodiments, the Grp94 inhibitors of Formula (III) have one ofthe Formula of Table 6, wherein each substituent is as defined above anddescribed in classes and subclasses herein, both singly and incombination.

TABLE 6 Formula Compound IIIA

IIIB

IIIC

IIID

IIIE

IIIF

IIIG

IIIH

IIII

IIIJ

IIIK

IIIL

IIIM

IIIN

IIIO

IIIP

IIIQ

IIIR

IIIS

IIIT

IIIU

IIIV

IIIW

IIIX

IIIY

IIIZ

IIIAA

IIIAB

IIIAC

IIIAD

IIIAE

IIIAF

IIIAG

IIIAH

IIIAI

IIIAJ

IIIAK

IIIAL

IIIAM

IIIAN

IIIAO

IIIAP

IIIAQ

IIIAR

IIIAS

IIIAT

wherein each R⁹ is R.

Illustrative compounds of Formula (III) are listed below in Table 7.

TABLE 7

and pharmaceutically acceptable salts thereof, where: R⁷ X¹⁰ X¹¹ X¹² X¹³(H₂C)₃—≡ Cl Cl Cl CH (H₂C)₃—≡ H H H CH (H₂C)₃—≡ H Cl H CH (H₂C)₃—≡ H HCl CH (H₂C)₃—≡ Cl H Cl CH (H₂C)₃—≡ Cl Cl H CH (H₂C)₃—≡ Cl Cl Cl N(H₂C)₃—≡ H H H N (H₂C)₃—≡ H Cl H N (H₂C)₃—≡ H H Cl N (H₂C)₃—≡ Cl H Cl N(H₂C)₃—≡ Cl Cl H N (CH₂)₃—NHCH(CH₃)₂ Cl Cl Cl CH (CH₂)₃—NHCH(CH₃)₂ H H HCH (CH₂)₃—NHCH(CH₃)₂ H Cl H CH (CH₂)₃—NHCH(CH₃)₂ H H Cl CH(CH₂)₃—NHCH(CH₃)₂ Cl H Cl CH (CH₂)₃—NHCH(CH₃)₂ Cl Cl H CH(CH₂)₃—NHCH(CH₃)₂ Cl Cl Cl N (CH₂)₃—NHCH(CH₃)₂ H H H N (CH₂)₃—NHCH(CH₃)₂H Cl H N (CH₂)₃—NHCH(CH₃)₂ H H Cl N (CH₂)₃—NHCH(CH₃)₂ Cl H Cl N(CH₂)₃—NHCH(CH₃)₂ Cl Cl H N

5.3.4 Grp94 Inhibitors of Formula (IV)

In one aspect, the disclosure encompasses purine-scaffold compounds thatare represented schematically in Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

(a) Y is —C(R^(Y))₂—, —S—, —NR—, —O—,

(b) each of Z¹, Z³, Z⁹, Z¹⁰, Z¹¹ and Z¹² are independently —CH— or —N—;

(c) Z² is —N— or —CR¹⁰—, wherein R¹⁰ is H or unsubstituted orsubstituted —(C₁-C₆)aliphatic;

(d) each of X⁸ and X⁹ are independently —CH—, —S—, —N—, or —O—;

(e) X¹ is —H, -halo, —N(R)₂, —OR, —CN, or unsubstituted or substituted—(C₁-C₆)aliphatic;

(f) R⁷ is —(C₁-C₆)aliphatic-N⁺—(R²)(R³)(R⁴), —(C₁-C₆)aliphatic-N—R³R⁴,—(C₁-C₆)aliphatic-C(═O)N—R³R⁴, —(C₁-C₆)aliphatic-R³R⁴,—(C₁-C₆)aliphatic-R²R³R⁴, —(C₁-C₆)aliphatic-N—CR²R³R⁴,—(C₁-C₆)aliphatic-C(halo)₃, —(C₁-C₆)aliphatic-alkenyl,—(C₁-C₆)aliphatic-alkynyl, —(C₁-C₆)aliphatic-(C₃-C₈)cycloalkyl,—(C₁-C₆)aliphatic-(C₃-C₈)heterocycloalkyl, —(C₁-C₆)aliphatic-phenyl,—(C₁-C₆)aliphatic-(5 or 6-membered)heteroaryl, —(C₁-C₆)aliphatic-cyano,with the proviso that when all of R²-R⁴ are present the compound furthercomprises a pharmaceutically acceptable counter ion;

(g) R² and R³ are independently hydrogen, —N(R)₂, —CH₂CH(OH)R⁴,—CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴, or unsubstituted or substituted—(C₁-C₆)aliphatic, or R³ and R⁴ form an unsubstituted or substituted 3-to 7-membered heterocyclic ring when taken together with the nitrogen towhich they are attached;

(h) R⁴ is hydrogen, halogen, or unsubstituted or substituted—(C₁-C₆)aliphatic;

(i) each R⁸ is independently —H, -halo, —N(R)₂, —OR, —CN, orunsubstituted or substituted —(C₁-C₆)aliphatic;

(j) R⁹ is —H, (C₁-C₆)aliphatic-cycloalkyl,—(C₁-C₆)aliphatic-heterocycloalkyl, —(C₁-C₆)aliphatic-aryl,—(C₁-C₆)aliphatic-heteroaryl, or —(C₁-C₆)aliphatic-cyano, wherein eachcycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted orsubstituted, with the proviso that R⁹ is absent when X⁹ is —S— or —O—;

(k) each R^(Y) is independently R, —OR, or halo;

(l) a is an integer selected from 0, 1 and 2; and

(m) each R is independently hydrogen, unsubstituted C₁₋₆ aliphatic, orC₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂; and

wherein each substituted group is substituted with one or more groupsselected from halo, —N(R)₂, —OR, —CN, oxo, unsubstituted C₁₋₆ aliphatic,or C₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂.

In some embodiments, a compound of formula (IV) or pharmaceuticallyacceptable salt thereof is defined wherein:

(a) Y is —CH₂—, —S—, —N—, —O—,

(b) each of Z¹, ³, Z⁹, Z¹⁰, Z¹¹ and Z¹² are independently —CH— or —N—;

(c) Z² is —CH—, —N—, or —CR¹⁰—, wherein R¹⁰ is —(C₁-C₆)alkyl;

(d) each of X⁸ and X⁹ are independently —CH—, —S—, —N—, or —O—;

(e) X¹ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, or—OCH₂(halo);

(f) R⁷ is —(CH₂)_(m)—N⁺—(R²)(R³)(R⁴), —(CH₂)_(m)—N—R³R⁴,—(CH₂)_(m)—C(═O)N—R³R⁴, —(CH₂)_(m)—R³R⁴, —(CH₂)_(m)—C(halo)₃,—(CH₂)_(m)-alkenyl, (CH₂)_(m)-alkenyl-CH₃, —(CH₂)_(m)-alkynyl,(CH₂)_(m)-alkynyl-CH₃, (CH₂)_(m)—(C₃-C₈)cycloalkyl,—(CH₂)_(m)—(C₃-C₈)heterocycloalkyl, —(CH₂)_(m)-phenyl, —(CH₂)_(m)-(5 or6-membered)heteroaryl, —(CH₂)_(m)-cyano, where m is 1, 2, 3, 4 or 5 andwhere the cyloalkyl, heterocycle or phenyl is unsubstituted orsubstituted with one or more X¹ groups, with the proviso that when allof R²-R⁴ are present the compound further comprises a pharmaceuticallyacceptable counter ion;

(g) R² and R³ are independently hydrogen, methyl, ethyl, ethenyl,ethynyl, propyl, butyl, pentyl, hexyl, isopropyl, t-butyl, isobutyl,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —CH₂C(halo)₃, —CHCH(halo)₂,CHCH₂(halo), —CH₂OH, —CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH₂CH(CH₃)OH,—C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH or R² and R³ form an unsubstituted orsubstituted aziridine, azetidine, pyrrolidine, piperazine, or piperidinering when taken together with the nitrogen to which they are attached;

(h) R⁴ is hydrogen, methyl, ethyl, isopropyl, t-butyl, isobutyl, or—C(halo)₃;

(i) R⁸ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, and—OCH₂(halo);

(j) R⁹ is —H, (CH₂)n-cycloalkyl, —(CH₂)n-heterocycloalkyl, —(CH₂)n-aryl,—(CH₂)n-heteroaryl, or —(CH₂)-cyano, wherein said cycloalkyl,heterocycloalkyl, aryl or heteroaryl is optionally substituted with oneor more X¹ groups;

(k) a is an integer selected from 0, 1 and 2; and

(l) n is an integer selected from 1, 2, 3 or 4.

In certain embodiments, Y is —S—, —CH₂—, or

in another embodiment, Y is S or

In another embodiment, Y is —S— or —CH₂—. In another embodiment, Y is—S— or —O—. In another embodiment, Y is —S—. In another embodiment, Y is—CH₂—. In another embodiment, Y is

In some embodiments, Y is —C(R^(Y))₂—, wherein each R^(Y) isindependently hydrogen, —OH, or halo.

In certain embodiments, Z¹ and Z² are —N—. In other embodiments, Z¹ is—N— and Z² is —C—.

In certain embodiments, R⁷ is —(CH₂)_(m)—N—(R³)(R⁴). In one suchembodiment, R¹ is —(CH₂)₂—N—(R³)(R⁴). In another such embodiment, R¹ is—(CH₂)₃—N—(R³)(R⁴). In another such embodiment, R⁷ is—(CH₂)₂—N—(R³)(R⁴), R³ is H and R⁴ is isopropyl or isobutyl. In anothersuch embodiment, R⁷ is —(CH₂)₃—N—R³R⁴, R³ is H and R⁴ is isopropyl orisobutyl. In another such embodiment, R⁷ is —(CH₂)₂—N—R³R⁴, R³ is H andR⁴ is isopropyl. It will be understood, that in these embodiments, theamine functionality may exist as a free base or as an acid additionsalt. Acid addition salts can be prepared by addition of a suitableacid, as is well understood in the art. In particular embodiments, theacid addition salt may be a hydrochloride salt, a phosphate salt, asulfate salt, a lactate salt, a citrate salt, a succinate salt, abenzene sulfonic acid salt, a mesylate salt, a tartrate salt, alactobionate salt, a para-toluenesulfonic acid salt, or a fumaricacid-salt. In another embodiment, the acid addition salt is ahydrochloride salt or a sulfate salt. In another embodiment, the acidaddition salt is a hydrochloride salt. In another embodiment, the acidaddition salt is a sulfate salt. In another embodiment, the acidaddition salt is a phosphate salt. When prepared as an acid additionsalt, the purine-scaffold inhibitors are rendered water soluble.Solubility may be increased even further by production of higher ordersalts, particularly di-salts. For instance, in embodiments where Z₁ is—N—, the nitrogen is ionizable and can be converted to an acid additionsalt under strongly acidic conditions (e.g., pH of less than about 3).Accordingly, Grp94 inhibitors of the disclosure in which Z₁ is —N— andthe R⁷ group contains an amine functionality can be converted intodi-salts. In certain embodiments, the Grp94 inhibitors of the disclosurecan be in the form of a di-HCl salt.

In certain embodiments, R⁷ is —(CH₂)_(m)—CF₃. In one such embodiment, R⁷is —(CH₂)₃—CF₃.

In another such embodiment, R⁷ is —(CH₂)₄—CF₃. In another suchembodiment, R⁷ is —(CH₂)₂—CF₃.

In another embodiment, R⁷ is —(CH₂)₃-alkynyl. In another embodiment, R⁷is —(CH₂)₃—CCH. In another embodiment, R⁷ is —(CH₂)₄-alkynyl. In anotherembodiment, R⁷ is —(CH₂)_(m)-cyano.

In certain embodiments, R⁹ is —(CH₂)-aryl. In one such embodiment, R⁹ is—(CH₂)-aryl. In another such embodiment, R⁹ is an unsubstituted benzylgroup. In another such embodiment, R⁹ is a substituted benzyl group. Inanother such embodiment, R⁹ is a para-substituted substituted benzylgroup. In another such embodiment, R⁹ is a para-methoxy substitutedbenzyl group.

In other embodiments, the Grp94 inhibitors of Formula (IV) have one ofthe Formula of Table 8, wherein each substituent is as defined above anddescribed in classes and subclasses herein, both singly and incombination.

TABLE 8 Formula Compound IVA

IVB

IVC

IVD

IVE

IVF

IVG

IVH

IVI

IVJ

IVK

IVL

IVM

IVN

IVO

IVP

Illustrative compounds of Formula (IV) are listed below in Tables 9.

TABLE 9

and pharmaceutically acceptable salts thereof, where: R⁷ X¹ Z⁹ R⁸(H₂C)₃—≡ H CH H (H₂C)₃—≡ H N H (H₂C)₃—≡ H CH Br (H₂C)₃—≡ H N Br (H₂C)₃—≡OCH₃ CH H (H₂C)₃—≡ OCH₃ N H (H₂C)₃—≡ OCH₃ CH Br (H₂C)₃—≡ OCH₃ N Br(CH₂)₃—NHCH(CH₃)₂ H CH H (CH₂)₃—NHCH(CH₃)₂ H N H (CH₂)₃—NHCH(CH₃)₂ H CHBr (CH₂)₃—NHCH(CH₃)₂ H N Br (CH₂)₃—NHCH(CH₃)₂ OCH₃ CH H(CH₂)₃—NHCH(CH₃)₂ OCH₃ N H (CH₂)₃—NHCH(CH₃)₂ OCH₃ CH Br(CH₂)₃—NHCH(CH₃)₂ OCH₃ N Br

5.3.5 Grp94 Inhibitors of Formula (V)

In one aspect, the disclosure encompasses purine-scaffold compounds thatare substituted at the 8-position with a linker group bonded to aryl orheteroaryl group and are further substituted at the N-3 position. Suchcompounds are represented schematically in Formula (V):

or a pharmaceutically acceptable salt thereof, wherein:

(a) Y is —C(R^(Y))₂-, —S—, —NR—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—;

(c) Z² is —N— or —CR¹⁰—, wherein R¹⁰ is H or unsubstituted orsubstituted —(C₁-C₆)aliphatic;

(d) each of Z⁴, Z⁵, Z⁶, Z⁷ and Z⁸ are independently —C— or —N—, with theproviso that no three consecutive Z⁴ through Z⁸ are N;

(e) X¹ is —H, -halo, —N(R)₂, —OR, —CN, or unsubstituted or substituted—(C₁-C₆)aliphatic;

(f) each of X⁴, X⁵, and X⁶ are independently —H, -halo, —SR, —N(R)₂,—OR, —CN, —NO₂, —CN, —C(O)R, —C(O)₂R, —S(O)R, —S(O)₂R, —C(O)N(R)₂,—SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)SO₂R, —OC(O)N(R)₂, unsubstituted orsubstituted —(C₁-C₆)aliphatic, or an unsubstituted or substituted groupselected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and(5- or 6-membered)heterocyclic aromatic or heterocyclic non-aromaticgroup; with the provisos that at least one of X², X⁴ and X⁵ is —H andthat X² is absent when Z⁴ is —N—, X³ is absent when Z⁵ is —N—, X⁴ isabsent when Z⁶ is —N— and X⁵ is absent when Z⁷ is —N—;

(g) each of X² and X³ are independently selected from

-   -   (1) —H, -halo, —SR, —N(R)₂, —OR, —CN, —NO₂, —CN, —C(O)R,        —C(O)₂R, —S(O)R, —S(O)₂R, —C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R,        —N(R)C(O)R, —N(R)SO₂R, —OC(O)N(R)₂, unsubstituted or substituted        —(C₁-C₆)aliphatic, or an unsubstituted or substituted group        selected from (5- or 6-membered)aryl, (5- or        6-membered)arylalkyl, and (5- or 6-membered)heterocyclic        aromatic or heterocyclic non-aromatic group; or    -   (2) X² and X³ taken together form a fused benzo or fused (5- or        6-membered) heteroaryl that may be substituted with one or more        R⁸ groups;

(h) R⁷ is —(C₁-C₆)aliphatic-N⁺—(R²)(R³)(R⁴), —(C₁-C₆)aliphatic-N—R³R⁴,—(C₁-C₆)aliphatic-C(═O)N—R³R⁴, —(C₁-C₆)aliphatic-R³R⁴,—(C₁-C₆)aliphatic-R²R³R⁴, —(C₁-C₆)aliphatic-N—CR²R³R⁴,—(C₁-C₆)aliphatic-C(halo)₃, —(C₁-C₆)aliphatic-alkenyl,—(C₁-C₆)aliphatic-alkynyl, —(C₁-C₆)aliphatic-(C₃-C₈)cycloalkyl,—(C₁-C₆)aliphatic-(C₃-C₈)heterocycloalkyl, —(C₁-C₆)aliphatic-phenyl,—(C₁-C₆)aliphatic-(5 or 6-membered)heteroaryl, —(C₁-C₆)aliphatic-cyano,with the proviso that when all of R²-R⁴ are present the compound furthercomprises a pharmaceutically acceptable counter ion;

(i) R² and R³ are independently hydrogen, —N(R)₂, —CH₂CH(OH)R⁴,—CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴, or unsubstituted or substituted—(C₁-C₆)aliphatic, or R³ and R⁴ form an unsubstituted or substituted 3-to 7-membered heterocyclic ring when taken together with the nitrogen towhich they are attached;

(j) R⁸ is —H, -halo, —SR, —N(R)₂, —OR, —CN, —NO₂, —CN, —C(O)R, —C(O)₂R,—S(O)R, —S(O)₂R, —C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)SO₂R,—OC(O)N(R)₂, unsubstituted or substituted —(C₁-C₆)aliphatic, or anunsubstituted or substituted group selected from (5- or 6-membered)aryl,(5- or 6-membered)arylalkyl, and (5- or 6-membered)heterocyclic aromaticor heterocyclic non-aromatic group;

(k) each R^(Y) is independently R, —OR, or halo;

(l) R⁴ is hydrogen, halogen, or unsubstituted or substituted—(C₁-C₆)aliphatic; and

(m) each R is independently hydrogen, unsubstituted C₁₋₆ aliphatic, orC₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂;

wherein each substituted group is substituted with one or more groupsselected from halo, —N(R)₂, —OR, —CN, oxo, unsubstituted C₁₋₆ aliphatic,or C₁₋₆ aliphatic substituted with halo, —OH, —CN, or —NH₂.

In some embodiments, a compound of formula (V) or pharmaceuticallyacceptable salt thereof is defined wherein:

(a) Y is —CH₂—, —S—, —N—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—;

(c) Z² is —CH—, —N—, or —CR¹⁰—, wherein R¹⁰ is —(C₁-C₆)alkyl;

(d) each of Z⁴, Z⁵, Z⁶, Z⁷ and Z⁸ are independently —CH— or —N—, withthe proviso that no three consecutive Z⁴ through Z⁸ are N;

(e) X¹ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, or—OCH₂(halo);

(f) each of X⁴, X⁵, and X⁶ are independently —H, -halo, —NH₂, —CN,—(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, —OCH₂(halo), pyridyl, furyl,thiophenyl, pyrrolyl, oxazolyl, imidazolyl, thiazolidinyl, thiadiazolyl,thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, triazinyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl,tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, ortetrahydrothiopyranyl

(g) each of X² and X³ are independently selected from

-   -   (1) —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,        —C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂,        —OCH₂(halo), pyridyl, furyl, thiophenyl, pyrrolyl, oxazolyl,        imidazolyl, thiazolidinyl, thiadiazolyl, thiazolyl, isoxazolyl,        pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl,        morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,        piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl,        tetrahydropyridinyl, tetrahydropyrimidinyl,        tetrahydrothiophenyl, or tetrahydrothiopyranyl; and    -   (2) X² and X³ taken together form a fused benzo or fused (5- or        6-membered) heteroaryl that may be substituted with one or more        R⁸ groups;

(h) R⁷ is —(CH₂)_(m)—N⁺—(R²)(R³)(R⁴), —(CH₂)_(m)—N—R³R⁴,—(CH₂)_(m)—C(═O)N—R³R⁴, —(CH₂)_(m)—C(halo)₃, —(CH₂)_(m)-alkenyl,(CH₂)_(m)-alkenyl-CH₃, —(CH₂)_(m)-alkynyl, (CH₂)_(m)-alkynyl-CH₃,(CH₂)_(m)—(C₃-C₈)cycloalkyl, —(CH₂)_(m)—(C₃-C₈)heterocycloalkyl,—(CH₂)_(m)-phenyl, —(CH₂)_(m)-(5 or 6-membered)heteroaryl,—(CH₂)_(m)-cyano, where m is 1, 2, 3, 4 or 5 and where the cyloalkyl,heterocycle or phenyl is unsubstituted or substituted with one or moreX¹ groups, with the proviso that when all of R²-R⁴ are present thecompound further comprises a pharmaceutically acceptable counter ion;

(i) R² and R³ are independently hydrogen, methyl, ethyl, ethenyl,ethynyl, propyl, butyl, pentyl, hexyl, isopropyl, t-butyl, isobutyl,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —CH₂C(halo)₃, —CHCH(halo)₂,CHCH₂(halo), —CH₂OH, —CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH₂CH(CH₃)OH,—C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH or R² and R³ form an unsubstituted orsubstituted aziridine, azetidine, pyrrolidine, piperazine, or piperidinering when taken together with the nitrogen to which they are attached;

(j) R⁴ is hydrogen, methyl, ethyl, isopropyl, t-butyl, isobutyl, or—C(halo)₃; and

(k) R⁸ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH,—C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, and—OCH₂(halo), and X³, X⁴, X⁵, and X⁶ are independently selected from —H,-halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃,—CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, —OCH₂(halo), pyridyl,furyl, phenyl, benzyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl,thiazolidinyl, thiadiazolyl, thiazolyl, isoxazolyl, pyrazolyl,isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl,2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, or terrahydrothiopyranyl.

In one embodiment, Z¹ and Z³ are —N—. In another embodiment, Z¹ is —N—and Z³ is —C—. In another embodiment, Z¹ is —C— and Z³ is —N—.

In another embodiment, Z⁴, Z⁵, Z⁶, Z⁷ and Z⁸ are —C—. In anotherembodiment, Z⁴ is —N— and Z⁵, Z⁶, Z⁷ and Z⁸ are —C—. In anotherembodiment, Z⁵ is —N— and Z⁴, Z⁶, Z⁷ and Z⁸ are —C—. In anotherembodiment, Z⁶ is —N— and Z⁴, Z⁵, Z⁷ and Z⁸ are —C—. In anotherembodiment, Z⁷ is —N— and Z⁴, Z⁵, Z⁶ and Z⁸ are —C—. In anotherembodiment, Z⁸ is —N— and Z⁴, Z⁵, Z⁶ and Z⁷ are —C—. In anotherembodiment, Z⁷ and Z⁴ are —N— and Z⁵, Z⁶ and Z⁸ are —C—. In anotherembodiment, Z⁵ and Z⁸ are —N— and Z⁴, Z⁶ and Z⁷ are —C—.

In another embodiment, Y is —S—, —CH₂—, or

In another embodiment, Y is S or

In another embodiment, Y is —S— or —CH₂—. In another embodiment, Y is—S— or —O—. In another embodiment, Y is —S—. In another embodiment, Y is—CH₂—. In another embodiment, Y is

In some embodiments, Y is —C(R^(Y))₂-, wherein each R^(Y) isindependently hydrogen, —OH, or halo.

In certain embodiment, R⁷ is —(CH₂)_(m)—N—(R³)(R⁴). In one suchembodiment, R¹ is —(CH₂)₂—N—(R³)(R⁴). In another such embodiment, R¹ is—(CH₂)₃—N—(R³)(R⁴). In another such embodiment, R⁷ is—(CH₂)₂—N—(R³)(R⁴), R³ is H and R⁴ is isopropyl or isobutyl. In anothersuch embodiment, R⁷ is —(CH₂)₃—N—(R³)(R⁴), R³ is H and R⁴ is isopropylor isobutyl. In another such embodiment, R⁷ is —(CH₂)₃—N—(R³)(R⁴), R³ isH and R⁴ is isopropyl. In another such embodiment, R⁷ is—(CH₂)₃—N—(R³)(R⁴), R³ is H and R⁴ is isobutyl. It will be understood,that in these embodiments, the amine functionality may exist as a freebase or as an acid addition salt. Acid addition salts can be prepared byaddition of a suitable acid, as is well understood in the art. Inparticular embodiments, the acid addition salt may be a hydrochloridesalt, a phosphate salt, a sulfate salt, a lactate salt, a citrate salt,a succinate salt, a mesylate salt, a tartrate salt, a lactobionate salt,a benzene sulfonic acid salt, a para-toluenesulfonic acid salt, or afumaric acid-salt. In another embodiment, the acid addition salt is ahydrochloride salt or a sulfate salt. In another embodiment, the acidaddition salt is a hydrochloride salt. In another embodiment, the acidaddition salt is a sulfate salt. In another embodiment, the acidaddition salt is a phosphate salt. When prepared as an acid additionsalt, the purine-scaffold inhibitors are rendered water soluble.Solubility may be increased even further by production of higher ordersalts, particularly di-salts. For instance, in embodiments where Z₁ is—N—, the nitrogen is ionizable and can be converted to an acid additionsalt under strongly acidic conditions (e.g., pH of less than 3).Accordingly, Grp94 inhibitors of the disclosure in which Z₁ is —N— andthe R⁷ group contains an amine functionality can be converted intodi-salts. In certain embodiments, the Grp94 inhibitors of the disclosurecan be in the form of a di-HCl salt.

In certain embodiments, R⁷ is —(CH₂)_(m)—CF₃. In one such embodiment, R⁷is —(CH₂)₃—CF₃. In another such embodiment, R⁷ is —(CH₂)₄—CF₃.

In another embodiment, R⁷ is —(CH₂)₃-alkenyl. In another embodiment, R⁷is —(CH₂)₃—CCH. In another embodiment, R⁷ is —(CH₂)₄-alkenyl.

In some embodiments, R⁷ is CH₂CCCH₃.

In another embodiment, R⁷ is —(CH₂)₂-alkynyl. In another embodiment, R⁷is —(CH₂)₃-alkynyl. In another embodiment, R⁷ is —(CH₂)₃—CCH. In anotherembodiment, R⁷ is —(CH₂)₄-alkynyl. In another embodiment, R⁷ is—(CH₂)₄—CCH. In another embodiment, R⁷ is —(CH₂)_(m)-cyano.

In some embodiments, R⁷ is benzyl.

In another embodiment, X¹ is —H. In another embodiment, X¹ is a halogenatom. In another embodiment, X¹ is —F. In another embodiment, X¹ is —Cl.

In another embodiment, X² is a halogen atom and X³, X⁴, X⁵ and X⁶ arehydrogen. In another embodiment X² is —Cl and X³, X⁴, X⁵ and X⁶ arehydrogen. In another embodiment X² is —OCH₃ and X³, X⁴, X⁵ and X⁶ arehydrogen. In another embodiment X² is —OCF₃ and X³, X⁴, X⁵ and X⁶ arehydrogen.

In another embodiment, X⁴ is a halogen atom and X², X³, X⁵ and X⁶ arehydrogen. In another embodiment X⁴ is —Cl and X², X³, X⁵ and X⁶ arehydrogen. In another embodiment X⁴ is —OCH₃ and X², X³, X⁵ and X⁶ arehydrogen. In another embodiment X⁴ is —OCF₃ and X², X³, X⁵ and X⁶ arehydrogen.

In certain embodiments, Z⁴ and Z⁶ are —C—, X² and X⁴ are independentlyselected from —H, -halo, —(C₁-C₃)alkyl and —O(C₁-C₃)alkyl and Z⁵, Z⁷ andZ⁸ are either an unsubstituted carbon or a nitrogen atom. In one suchembodiment, at least one of X² and X⁴ are -halo. In another suchembodiment, both X² and X⁴ are —Cl. In another such embodiment, at leastone of X² and X⁴ are alkyl groups. In another such embodiment, both X²and X⁴ are —CH₃. In another such embodiment, at least one of X² and X⁴are —OCH₃. In another such embodiment, at least one of X² and X⁴ are—CF₃.

In certain embodiments, Z⁴ and Z⁷ are —C—, X² and X⁵ are independentlyselected from —H, -halo, —(C₁-C₃)alkyl, —(C₁-C₃)haloalkyl,—(C₁-C₃)haloalkyl, and —O(C₁-C₃)alkyl and Z⁵, Z⁶ and Z⁸ are either anunsubstituted carbon or a nitrogen atom. In one such embodiment, atleast one of X² and X⁵ are halogen atoms. In another such embodiment,both X² and X⁵ are —Cl. In another such embodiment, at least one of X²and X⁴ are alkyl groups. In another such embodiment, both X² and X⁵ are—CH₃. In another such embodiment, at least one of X² and X⁴ are —CF₃

In certain embodiments, Z⁵ and Z⁷ are —C—, X³ and X⁵ are independentlyselected from —H, -halo, —(C₁-C₃)alkyl, —(C₁-C₃)haloalkyl,—O(C₁-C₃)haloalkyl, and —O(C₁-C₃)alkyl and Z⁴, Z⁶ and Z⁸ are either anunsubstituted carbon or a nitrogen atom. In one such embodiment, atleast one of X³ and X⁵ are halogen atoms. In another such embodiment,both X³ and X⁵ are —Cl. In another such embodiment, at least one of X³and X⁵ are alkyl groups. In another such embodiment, both X³ and X⁵ are—CH₃. In another such embodiment, at least one of X³ and X⁵ are —CF₃.

In some embodiments, X³ and X⁴ are halo and X², X⁵, and X⁶ are hydrogen.

In some embodiments, X², X⁴ and X⁵ are halo and X³ and X⁶ are hydrogen.In some embodiments, X², X³ and X⁵ are halo and X⁴ and X⁶ are hydrogen.In some embodiments, X², X³ and X⁴ are halo and X⁵ and X⁶ are hydrogen.

In some embodiments, X², X⁴, and X⁶ are methyl and X³ and X⁵ arehydrogen.

In another embodiment, X² and X³ taken together form a fused benzo. Inanother embodiment, X² and X³ taken together form a substituted orunsubstituted fused pyridyl.

In some embodiments, the Grp94 inhibitors of Formula (V) are of Formula(Va):

or a pharmaceutically acceptable salt thereof, wherein each of X¹, R⁷,Y, X³, and X⁵ is as defined above and described in classes andsubclasses herein, both singly and in combination.

In some embodiments, the Grp94 inhibitors of Formula (V) are of Formula(Vb):

or a pharmaceutically acceptable salt thereof, wherein R⁷ is as definedabove where i) the —(C₁-C₆)aliphatic group attached to the ring nitrogenis —(CH₂)₃— or ii) m is 3; and each of X¹, Y, X², X³, X⁴, X⁵ and X⁶ isas defined above and described in classes and subclasses herein, bothsingly and in combination.

In other embodiments, the Grp94 inhibitors of Formula (V) have one ofthe Formula of Table 10, wherein each substituent is as defined aboveand described in classes and subclasses herein, both singly and incombination.

TABLE 10 Formula Compound VA

VB

VC

VD

VE

VF

VG

VH

VV

VJ

VK

VL

VM

VN

VO

VP

VQ

VR

VS

VT

VU

VV

VW

VX

VY

VZ

VAA

VAB

VAC

VAD

VAE

VAF

TABLE 11

and pharmaceutically acceptable salts thereof, where: m R⁴ Y X² X³ X⁴ X⁵3 CH(CH₃)₂ S H Cl H H 3 CH(CH₃)₂ S H H H Cl 3 CH(CH₃)₂ S H H Cl H 3CH(CH₃)₂ S H Cl H Cl 3 CH(CH₃)₂ S H Cl Cl Cl 3 CH(CH₃)₂ O H Cl H H 3CH(CH₃)₂ O H H H Cl 3 CH(CH₃)₂ O H H Cl H 3 CH(CH₃)₂ O H Cl H Cl 3CH(CH₃)₂ O H Cl Cl Cl 3 CH(CH₃)₂ CH₂ H Cl H H 3 CH(CH₃)₂ CH₂ H H H Cl 3CH(CH₃)₂ CH₂ H H Cl H 3 CH(CH₃)₂ CH₂ H Cl H Cl 3 CH(CH₃)₂ CH₂ H Cl Cl Cl3 CH(CH₃)₂ C═O H H H Cl 3 CH(CH₃)₂ C═O H H Cl H 3 CH(CH₃)₂ C═O H Cl H Cl3 CH(CH₃)₂ C═O H Cl Cl Cl 3 CH(CH₃)₂ S H Cl H H 3 CH(CH₃)₂ S H H H Cl 3CH(CH₃)₂ S H H Cl H 3 CH(CH₃)₂ S H Cl H Cl 3 CH(CH₃)₂ S H Cl Cl Cl 3CH(CH₃)₂ O H Cl H H 3 CH(CH₃)₂ O H H H Cl 3 CH(CH₃)₂ O H H Cl H 3CH(CH₃)₂ O H Cl H Cl 3 CH(CH₃)₂ O H Cl Cl Cl 3 CH(CH₃)₂ CH₂ H Cl H H 3CH(CH₃)₂ CH₂ H H H Cl 3 CH(CH₃)₂ CH₂ H H Cl H 3 CH(CH₃)₂ CH₂ H Cl H Cl 3CH(CH₃)₂ CH₂ H Cl Cl Cl 3 CH(CH₃)₂ C═O H H H Cl 3 CH(CH₃)₂ C═O H H Cl H3 CH(CH₃)₂ C═O H Cl H Cl 3 CH(CH₃)₂ C═O H Cl Cl Cl 2 CH₂CH(OH)CH₃ S H ClH H 2 CH₂CH(OH)CH₃ S H H H Cl 2 CH₂CH(OH)CH₃ S H H Cl H 2 CH₂CH(OH)CH₃ SH Cl H Cl 2 CH₂CH(OH)CH₃ S H Cl Cl Cl 2 C(CH₃)₂CH₂OH S H Cl H H 2C(CH₃)₂CH₂OH S H H H Cl 2 C(CH₃)₂CH₂OH S H H Cl H 2 C(CH₃)₂CH₂OH S H ClH Cl 2 C(CH₃)₂CH₂OH S H Cl Cl Cl 2 CH₂CHF₂ S H Cl H H 2 CH₂CHF₂ S H H HCl 2 CH₂CHF₂ S H H Cl H 2 CH₂CHF₂ S H Cl H Cl 2 CH₂CHF₂ S H Cl Cl Cl 3CH₂C(CH₃)₂ S H Cl H H 3 CH₂C(CH₃)₂ S H H H Cl 3 CH₂C(CH₃)₂ S H H Cl H 3CH₂C(CH₃)₂ S H Cl H Cl 3 CH₂C(CH₃)₂ S H Cl Cl Cl 3 CH(CH3)₂ S H CH₃ H H3 CH(CH3)₂ S H H H CH₃ 3 CH(CH3)₂ S H H CH₃ H 3 CH(CH3)₂ S H CH₃ H CH₃ 3CH(CH3)₂ S H CH₃ CH₃ CH₃ 3 CH(CH3)₂ O H CH₃ H H 3 CH(CH3)₂ O H H H CH₃ 3CH(CH3)₂ O H H CH₃ H 3 CH(CH3)₂ O H CH₃ H CH₃ 3 CH(CH3)₂ O H CH₃ CH₃ CH₃3 CH(CH3)₂ CH₂ H CH₃ H H 3 CH(CH3)₂ CH₂ H H H CH₃ 3 CH(CH3)₂ CH₂ H H CH₃H 3 CH(CH3)₂ CH₂ H CH₃ H CH₃ 3 CH(CH3)₂ CH₂ H CH₃ CH₃ CH₃ 3 CH(CH3)₂ C═OH H H CH₃ 3 CH(CH3)₂ C═O H H CH₃ H 3 CH(CH3)₂ C═O H CH₃ H CH₃ 3 CH(CH3)₂C═O H CH₃ CH₃ CH₃ 3 CH(CH3)₂ S H CH₃ H H 3 CH(CH3)₂ S H H H CH₃ 3CH(CH3)₂ S H H CH₃ H 3 CH(CH3)₂ S H CH₃ H CH₃ 3 CH(CH3)₂ S H CH₃ CH₃ CH₃3 CH(CH3)₂ O H CH₃ H H 3 CH(CH3)₂ O H H H Cl 3 CH(CH3)₂ O H H CH₃ H 3CH(CH3)₂ O H CH₃ H CH₃ 3 CH(CH3)₂ O H CH₃ CH₃ CH₃ 3 CH(CH3)₂ CH₂ H CH₃ HH 3 CH(CH3)₂ CH₂ H H H CH₃ 3 CH(CH3)₂ CH₂ H H CH₃ H 3 CH(CH3)₂ CH₂ H CH₃H CH₃ 3 CH(CH3)₂ CH₂ H CH₃ CH₃ CH₃ 3 CH(CH3)₂ C═O H H H CH₃ 3 CH(CH3)₂C═O H H CH₃ H 3 CH(CH3)₂ C═O H CH₃ H CH₃ 3 CH(CH3)₂ C═O H CH₃ CH₃ CH₃ 2CH₂CH(OH)CH₃ S H CH₃ H H 2 CH₂CH(OH)CH₃ S H H H CH₃ 2 CH₂CH(OH)CH₃ S H HCH₃ H 2 CH₂CH(OH)CH₃ S H CH₃ H CH₃ 2 CH₂CH(OH)CH₃ S H CH₃ CH₃ CH₃ 2C(CH₃)₂CH₂OH S H CH₃ H H 2 C(CH₃)₂CH₂OH S H H H CH₃ 2 C(CH₃)₂CH₂OH S H HCH₃ H 2 C(CH₃)₂CH₂OH S H CH₃ H CH₃ 2 C(CH₃)₂CH₂OH S H CH₃ CH₃ CH₃ 2CH₂CHF₂ S H CH₃ H H 2 CH₂CHF₂ S H H H CH₃ 2 CH₂CHF₂ S H H CH₃ H 2CH₂CHF₂ S H CH₃ H CH₃ 2 CH₂CHF₂ S H CH₃ CH₃ CH₃ 3 CH₂C(CH₃)₂ S H CH₃ H H3 CH₂C(CH₃)₂ S H H H CH₃ 3 CH₂C(CH₃)₂ S H H CH₃ H 3 CH₂C(CH₃)₂ S H CH₃ HCH₃ 3 CH₂C(CH₃)₂ S H CH₃ CH₃ CH₃ 2 H₂C—C≡C S H CH₃ H H 2 H₂C—C≡C S H H HCH₃ 2 H₂C—C≡C S H H CH₃ H 2 H₂C—C≡C S H CH₃ H CH₃ 2 H₂C—C≡C S H CH₃ CH₃CH₃ 2 CH(CH₃)₂ S H Cl H H 2 CH₂CH₂OH S H H H Cl 2 CH₂CH(CH₃)OH S H H ClH 2 CH(CH₃)CH₂OH S Cl H H H 2 CH(CH₃)CH(CH₃)OH S H Br H H 2 C(CH₃)₂CH₂OHS H H H Br 2 CH₂C(CH₃)₂OH S H H Br H 2 CH₂CHF₂ S Br H H H 2 CH₂CF₃ S H IH H 2 CH₂CH(CH₃)₂ S H H H I 2 CH₂C(CH₃)₃ S H H I H 2 H₂C—C≡N S I H H H 3H₂C—C≡CH S H CH₃ H H 3 CH(CH₃)₂ S H H H CH₃ 3 CH₂CH₂OH S H H CH₃ H 3CH₂CH(CH₃)OH S CH₃ H H H 3 CH(CH₃)CH₂OH S H C₂H₅ H H 3 CH(CH₃)CH(CH₃)OHS H H H C₂H₅ 3 C(CH₃)₂CH₂OH S H H C₂H₅ H 3 CH₂C(CH₃)₂OH S C₂H₅ H H H 3CH₂CHF₂ S H i-C₃H₇ H H 3 CH₂CF₃ S H H H i-C₃H₇ 3 CH₂CH(CH₃)₂ S H Hi-C₃H₇ H 3 CH₂C(CH₃)₃ S i-C₃H₇ H H H 3 H₂C—C≡N O H Cl H H 2 H₂C—C≡CH O HH H Cl 2 CH(CH₃)₂ O H H Cl H 2 CH₂CH₂OH O Cl H H H 2 CH₂CH(CH₃)OH O H BrH H 2 CH(CH₃)CH₂OH O H H H Br 2 CH(CH₃)CH(CH₃)OH O H H Br H 2C(CH₃)₂CH₂OH O Br H H H 2 CH₂C(CH₃)₂OH O H I H H 2 CH₂CHF₂ O H H H I 2CH₂CF₃ O H H I H 2 CH₂CH(CH₃)₂ O I H H H 3 CH₂C(CH₃)₃ O H CH₃ H H 3H₂C—C≡N O H H H CH₃ 3 H₂C—C≡CH O H H CH₃ H 3 CH(CH₃)₂ O CH₃ H H H 3CH₂CH₂OH O H C₂H₅ H H 3 CH₂CH(CH₃)OH O H H H C₂H₅ 3 CH(CH₃)CH₂OH O H HC₂H₅ H 3 CH(CH₃)CH(CH₃)OH O C₂H₅ H H H 3 C(CH₃)₂CH₂OH O H I-C₃H₇ H H 3CH₂C(CH₃)₂OH O H H H i-C₃H₇ 3 CH₂CHF₂ O H H i-C₃H₇ H 3 CH₂CF₃ O i-C₃H₇ HH H 2 CH₂CH(CH₃)₂ S═O H Cl H H 2 CH₂C(CH₃)₃ S═O H H H Cl 2 H₂C—C≡N S═O HH Cl H 2 H₂C—C≡CH S═O Cl H H H 2 CH(CH₃)₂ S═O H Br H H 2 CH₂CH₂OH S═O HH H Br 2 CH₂CH(CH₃)OH S═O H H Br H 2 CH(CH₃)CH₂OH S═O Br H H H 2CH(CH₃)CH(CH₃)OH S═O H I H H 2 C(CH₃)₂CH₂OH S═O H H H I 2 CH₂C(CH₃)₂OHS═O H H I H 2 CH₂CHF₂ S═O I H H H 3 CH₂CF₃ S═O H CH₃ H H 3 CH₂CH(CH₃)₂S═O H H H CH₃ 3 CH₂C(CH₃)₃ S═O H H CH₃ H 3 H₂C—C≡N S═O CH₃ H H H 3H₂C—C≡CH S═O H C₂H₅ H H 3 CH(CH₃)₂ S═O H H H C₂H₅ 3 CH₂CH₂OH S═O H HC₂H₅ H 3 CH₂CH(CH₃)OH S═O C₂H₅ H H H 3 CH(CH₃)CH₂OH S═O H i-C₃H₇ H H 3CH(CH₃)₂ S═O H H H i-C₃H₇ 3 CH₂CH₂OH S═O H H i-C₃H₇ H 3 CH₂CH(CH₃)OH S═Oi-C₃H₇ H H H 2 CH(CH₃)CH₂OH O═S═O H Cl H H 2 CH(CH₃)CH(CH₃)OH O═S═O H HH Cl 2 C(CH₃)₂CH₂OH O═S═O H H Cl H 2 CH₂C(CH₃)₂OH O═S═O Cl H H H 2CH₂CHF₂ O═S═O H Br H H 2 CH₂CF₃ O═S═O H H H Br 2 CH₂CH(CH₃)₂ O═S═O H HBr H 2 CH₂C(CH₃)₃ O═S═O Br H H H 2 H₂C—C≡N O═S═O H I H H 2 H₂C—C≡CHO═S═O H H H I 2 CH(CH₃)₂ O═S═O H H I H 2 CH₂CH₂OH O═S═O I H H H 3CH₂CH(CH₃)OH O═S═O H CH₃ H H 3 CH(CH₃)CH₂OH O═S═O H H H CH₃ 3CH(CH₃)CH(CH₃)OH O═S═O H H CH₃ H 3 C(CH₃)₂CH₂OH O═S═O CH₃ H H H 3CH₂C(CH₃)₂OH O═S═O H C₂H₅ H H 3 CH₂CHF₂ O═S═O H H H C₂H₅ 3 CH₂CF₃ O═S═OH H C₂H₅ H 3 CH₂CH(CH₃)₂ O═S═O C₂H₅ H H H 3 CH₂C(CH₃)₃ O═S═O H i-C₃H₇ HH 3 H₂C—C≡N O═S═O H H H i-C₃H₇ 3 H₂C—C≡CH O═S═O H H i-C₃H₇ H 3 CH(CH₃)₂O═S═O i-C₃H₇ H H H 2 CH₂CH₂OH NH H Cl H H 2 CH₂CH(CH₃)OH NH H H H Cl 2CH(CH₃)CH₂OH NH H H Cl H 2 CH(CH₃)CH(CH₃)OH NH Cl H H H 2 C(CH₃)₂CH₂OHNH H Br H H 2 CH₂C(CH₃)₂OH NH H H H Br 2 CH₂CHF₂ NH H H Br H 2 CH₂CF₃ NHBr H H H 2 CH₂CH(CH₃)₂ NH H I H H 2 CH₂C(CH₃)₃ NH H H H I 2 H₂C—C≡N NH HH I H 2 H₂C—C≡CH NH I H H H 3 CH(CH₃)₂ NH H CH₃ H H 3 CH₂CH₂OH NH H H HCH₃ 3 CH₂CH(CH₃)OH NH H H CH₃ H 3 CH(CH₃)CH₂OH NH CH₃ H H H 3CH(CH₃)CH(CH₃)OH NH H C₂H₅ H H 3 C(CH₃)₂CH₂OH NH H H H C₂H₅ 3CH₂C(CH₃)₂OH NH H H C₂H₅ H 3 CH₂CHF₂ NH C₂H₅ H H H 3 CH₂CF₃ NH H i-C₃H₇H H 3 CH₂CH(CH₃)₂ NH H H H i-C₃H₇ 3 CH₂C(CH₃)₃ NH H H i-C₃H₇ H 3 H₂C—C≡NNH i-C₃H₇ H H H 2 H₂C—C≡CH C═O H Cl H H 2 CH(CH₃)₂ C═O H H H Cl 2CH₂CH₂OH C═O H H Cl H 2 CH₂CH(CH₃)OH C═O Cl H H H 2 CH(CH₃)CH₂OH C═O HBr H H 2 CH(CH₃)CH(CH₃)OH C═O H H H Br 2 C(CH₃)₂CH₂OH C═O H H Br H 2CH₂C(CH₃)₂OH C═O Br H H H 2 CH₂CHF₂ C═O H I H H 2 CH₂CF₃ C═O H H H I 2CH₂CH(CH₃)₂ C═O H H I H 2 CH₂C(CH₃)₃ C═O I H H H 3 H₂C—C≡N C═O H CH₃ H H3 H₂C—C≡CH C═O H H H CH₃ 3 CH(CH₃)₂ C═O H H CH₃ H 3 CH₂CH₂OH C═O CH₃ H HH 3 CH₂CH(CH₃)OH C═O H C₂H₅ H H 3 CH(CH₃)CH₂OH C═O H H H C₂H₅ 3CH(CH₃)CH(CH₃)OH C═O H H C₂H₅ H 3 C(CH₃)₂CH₂OH C═O C₂H₅ H H H 3CH₂C(CH₃)₂OH C═O H i-C₃H₇ H H 3 CH₂CHF₂ C═O H H H i-C₃H₇ 3 CH₂CF₃ C═O HH i-C₃H₇ H 3 CH₂CH(CH₃)₂ C═O i-C₃H₇ H H H 2 CH₂C(CH₃)₃ C═S H Cl H H 2H₂C—C≡N C═S H H H Cl 2 H₂C—C≡CH C═S H H Cl H 2 CH(CH₃)₂ C═S Cl H H H 2CH₂CH₂OH C═S H Br H H 2 CH₂CH(CH₃)OH C═S H H H Br 2 CH(CH₃)CH₂OH C═S H HBr H 2 CH(CH₃)CH(CH₃)OH C═S Br H H H 2 C(CH₃)₂CH₂OH C═S H I H H 2CH₂C(CH₃)₂OH C═S H H H I 2 CH₂CHF₂ C═S H H I H 2 CH₂CF₃ C═S I H H H 3CH₂CH(CH₃)₂ C═S H CH₃ H H 3 CH₂C(CH₃)₃ C═S H H H CH₃ 3 H₂C—C≡N C═S H HCH₃ H 3 H₂C—C≡CH C═S CH₃ H H H 3 CH(CH₃)₂ C═S H C₂H₅ H H 3 CH₂CH₂OH C═SH H H C₂H₅ 3 CH₂CH(CH₃)OH C═S H H C₂H₅ H 3 CH(CH₃)CH₂OH C═S C₂H₅ H H H 3CH(CH₃)CH(CH₃)OH C═S H i-C₃H₇ H H 3 C(CH₃)₂CH₂OH C═S H H H i-C₃H₇ 3CH₂C(CH₃)₂OH C═S H H i-C₃H₇ H 3 CH₂CHF₂ C═S H H H i-C₃H₇ 2 CH₂CF₃ CH₂ HCl H H 2 CH₂CH(CH₃)₂ CH₂ H H H Cl 2 CH₂C(CH₃)₃ CH₂ H H Cl H 2 H₂C—C≡NCH₂ Cl H H H 2 H₂C—C≡CH CH₂ H Br H H 2 CH(CH₃)₂ CH₂ H H H Br 2 CH₂CH₂OHCH₂ H H Br H 2 CH₂CH(CH₃)OH CH₂ Br H H H 2 CH(CH₃)CH₂OH CH₂ H I H H 2CH(CH₃)CH(CH₃)OH CH₂ H H H I 2 C(CH₃)₂CH₂OH CH₂ H H I H 2 CH₂C(CH₃)₂OHCH₂ I H H H 3 CH₂CHF₂ CH₂ H CH₃ H H 3 CH₂CF₃ CH₂ H H H CH₃ 3 CH₂CH(CH₃)₂CH₂ H H CH₃ H 3 CH₂C(CH₃)₃ CH₂ CH₃ H H H 3 H₂C—C≡N CH₂ H C₂H₅ H H 3H₂C—C≡CH CH₂ H H H C₂H₅ 3 CH(CH₃)₂ CH₂ H H C₂H₅ H 3 CH₂CH₂OH CH₂ C₂H₅ HH H 3 CH₂CH(CH₃)OH CH₂ H i-C₃H₇ H H 3 CH(CH₃)CH₂OH CH₂ H H H i-C₃H₇ 3CH(CH₃)CH(CH₃)OH CH₂ H H i-C₃H₇ H 3 C(CH₃)₂CH₂OH CH₂ i-C₃H₇ H H H 2CH₂C(CH₃)₂OH CH—OH H Cl H H 2 CH₂CHF₂ CH—OH H H H Cl 2 CH₂CF₃ CH—OH H HCl H 2 CH₂CH(CH₃)₂ CH—OH Cl H H H 2 CH₂C(CH₃)₃ CH—OH H Br H H 2 H₂C—C≡NCH—OH H H H Br 2 H₂C—C≡CH CH—OH H H Br H 2 CH(CH₃)₂ CH—OH Br H H H 2CH₂CH₂OH CH—OH H I H H 2 CH₂CH(CH₃)OH CH—OH H H H I 2 CH(CH₃)CH₂OH CH—OHH H I H 2 CH(CH₃)CH(CH₃)OH CH—OH I H H H 3 C(CH₃)₂CH₂OH CH—OH H CH₃ H H3 CH₂C(CH₃)₂OH CH—OH H H H CH₃ 3 CH₂CHF₂ CH—OH H H CH₃ H 3 CH₂CF₃ CH—OHCH₃ H H H 3 CH₂CH(CH₃)₂ CH—OH H C₂H₅ H H 3 CH₂C(CH₃)₃ CH—OH H H H C₂H₅ 3H₂C—C≡N CH—OH H H C₂H₅ H 3 H₂C—C≡CH CH—OH C₂H₅ H H H 3 CH(CH₃)₂ CH—OH Hi-C₃H₇ H H 3 CH₂CH₂OH CH—OH H H H i-C₃H₇ 3 CH₂CH(CH₃)OH CH—OH H H i-C₃H₇H 3 CH(CH₃)CH₂OH CH—OH i-C₃H₇ H H H 2 CH(CH₃)CH(CH₃)OH CH—F H Cl H H 2C(CH₃)₂CH₂OH CH—F H H H Cl 2 CH₂C(CH₃)₂OH CH—F H H Cl H 2 CH₂CHF₂ CH—FCl H H H 2 CH₂CF₃ CH—F H Br H H 2 CH₂CH(CH₃)₂ CH—F H H H Br 2 CH₂C(CH₃)₃CH—F H H Br H 2 H₂C—C≡N CH—F Br H H H 2 H₂C—C≡CH CH—F H I H H 2 CH(CH₃)₂CH—F H H H I 2 CH₂CH₂OH CH—F H H I H 2 CH₂CH(CH₃)OH CH—F I H H H 3CH(CH₃)CH₂OH CH—F H CH₃ H H 3 CH(CH₃)CH(CH₃)OH CH—F H H H CH₃ 3C(CH₃)₂CH₂OH CH—F H H CH₃ H 3 CH₂C(CH₃)₂OH CH—F CH₃ H H H 3 CH₂CHF₂ CH—FH C₂H₅ H H 3 CH₂CF₃ CH—F H H H C₂H₅ 3 CH₂CH(CH₃)₂ CH—F H H C₂H₅ H 3CH₂C(CH₃)₃ CH—F C₂H₅ H H H 3 H₂C—C≡N CH—F H i-C₃H₇ H H 3 H₂C—C≡CH CH—F HH H i-C₃H₇ 3 CH(CH₃)₂ CH—F H H i-C₃H₇ H 3 CH₂CH₂OH CH—F i-C₃H₇ H H H 2CH₂CH(CH₃)OH S H Cl H Cl 2 CH(CH₃)CH₂OH S Cl H Cl H 2 CH(CH₃)CH(CH₃)OH SCl H H Cl 2 C(CH₃)₂CH₂OH S H Br H Br 2 CH₂C(CH₃)₂OH S Br H Br H 2CH₂CHF₂ S Br H H Br 2 CH₂CF₃ S H I H I 2 CH₂CH(CH₃)₂ S I H I H 2CH₂C(CH₃)₃ S I H H I 2 H₂C—C≡N S H CH₃ H CH₃ 2 H₂C—C≡CH S CH₃ H CH₃ H 2CH(CH₃)₂ S CH₃ H H CH₃ 3 CH₂CH₂OH S H C₂H₅ H C₂H₅ 3 CH₂CH(CH₃)OH S C₂H₅H C₂H₅ H 3 CH(CH₃)CH2OH S C₂H₅ H H C₂H₅ 3 CH(CH₃)CH(CH₃)OH S H i-C₃H₇ Hi-C₃H₇ 3 C(CH₃)₂CH₂OH S i-C₃H₇ H i-C₃H₇ H 3 CH₂C(CH₃)₂OH S i-C₃H₇ H Hi-C₃H₇ 3 CH₂CHF₂ O H Cl H Cl 3 CH₂CF₃ O Cl H Cl H 3 CH₂CH(CH₃)₂ O Cl H HCl 3 CH₂C(CH₃)₃ O H Br H Br 3 H₂C—C≡N O Br H Br H 3 H₂C—C≡CH O Br H H Br2 CH(CH₃)₂ O H I H I 2 CH₂CH₂OH O I H I H 2 CH₂CH(CH₃)OH O I H H I 2CH(CH₃)CH2OH O H CH₃ H CH₃ 2 CH(CH₃)CH(CH₃)OH O CH₃ H CH₃ H 2C(CH₃)₂CH₂OH O CH₃ H H CH₃ 2 CH₂C(CH₃)₂OH O H C₂H₅ H C₂H₅ 2 CH₂CHF₂ OC₂H₅ H C₂H₅ H 2 CH₂CF₃ O C₂H₅ H H C₂H₅ 2 CH₂CH(CH₃)₂ O H i-C₃H₇ H i-C₃H₇2 CH₂C(CH₃)₃ O i-C₃H₇ H i-C₃H₇ H 2 H₂C—C≡N O i-C₃H₇ H H i-C₃H₇ 3H₂C—C≡CH S═O H Cl H Cl 3 CH(CH₃)₂ S═O Cl H Cl H 3 CH₂CH₂OH S═O Cl H H Cl3 CH₂CH(CH₃)OH S═O H Br H Br 3 CH(CH₃)CH2OH S═O Br H Br H 3CH(CH₃)CH(CH₃)OH S═O Br H H Br 3 C(CH₃)₂CH₂OH S═O H I H I 3 CH₂C(CH₃)₂OHS═O I H I H 3 CH₂CHF₂ S═O I H H I 3 CH₂CF₃ S═O H CH₃ H CH₃ 3 CH₂CH(CH₃)₂S═O CH₃ H CH₃ H 3 CH₂C(CH₃)₃ S═O CH₃ H H CH₃ 2 H₂C—C≡N S═O H C₂H₅ H C₂H₅2 H₂C—C≡CH S═O C₂H₅ H C₂H₅ H 2 CH(CH₃)₂ S═O C₂H₅ H H C₂H₅ 2 CH₂CH₂OH S═OH i-C₃H₇ H i-C₃H₇ 2 CH₂CH(CH₃)OH S═O i-C₃H₇ H i-C₃H₇ H 2 CH(CH₃)CH₂OHS═O i-C₃H₇ H H i-C₃H₇ 2 CH(CH₃)CH(CH₃)OH O═S═O H Cl H Cl 2 C(CH₃)₂CH₂OHO═S═O Cl H Cl H 2 CH₂C(CH₃)₂OH O═S═O Cl H H Cl 2 CH₂CHF₂ O═S═O H Br H Br2 CH₂CF₃ O═S═O Br H Br H 2 CH₂CH(CH₃)₂ O═S═O Br H H Br 3 CH₂C(CH₃)₃O═S═O H I H I 3 H₂C—C≡N O═S═O I H I H 3 H₂C—C≡CH O═S═O I H H I 3CH(CH₃)₂ O═S═O H CH₃ H CH₃ 3 CH₂CH₂OH O═S═O CH₃ H CH₃ H 3 CH₂CH(CH₃)OHO═S═O CH₃ H H CH₃ 3 CH(CH₃)CH₂OH O═S═O H C₂H₅ H C₂H₅ 3 CH(CH₃)CH(CH₃)OHO═S═O C₂H₅ H C₂H₅ H 3 C(CH₃)₂CH₂OH O═S═O C₂H₅ H H C₂H₅ 3 CH₂C(CH₃)₂OHO═S═O H i-C₃H₇ H i-C₃H₇ 3 CH₂CHF₂ O═S═O i-C₃H₇ H i-C₃H₇ H 3 CH₂CF₃ O═S═Oi-C₃H₇ H H i-C₃H₇ 2 CH₂CH(CH₃)₂ NH H Cl H Cl 2 CH₂C(CH₃)₃ NH Cl H Cl H 2H₂C—C≡N NH Cl H H Cl 2 H₂C—C≡CH NH H Br H Br 2 CH(CH₃)₂ NH Br H Br H 2CH₂CH₂OH NH Br H H Br 2 CH₂CH(CH₃)OH NH H I H I 2 CH(CH₃)CH₂OH NH I H IH 2 CH(CH₃)CH(CH₃)OH NH I H H I 2 C(CH₃)₂CH₂OH NH H CH₃ H CH₃ 2CH₂C(CH₃)₂OH NH CH₃ H CH₃ H 2 CH₂CHF₂ NH CH₃ H H CH₃ 3 CH₂CF₃ NH H C₂H₅H C₂H₅ 3 CH₂CH(CH₃)₂ NH C₂H₅ H C₂H₅ H 3 CH₂C(CH₃)₃ NH C₂H₅ H H C₂H₅ 3H₂C—C≡N NH H i-C₃H₇ H i-C₃H₇ 3 H₂C—C≡CH NH i-C₃H₇ H i-C₃H₇ H 3 CH(CH₃)₂NH i-C₃H₇ H H i-C₃H₇ 3 CH₂CH₂OH C═O H Cl H Cl 3 CH₂CH(CH₃)OH C═O Cl H ClH 3 CH(CH₃)CH₂OH C═O Cl H H Cl 3 CH(CH₃)CH(CH₃)OH C═O H Br H Br 3C(CH₃)₂CH₂OH C═O Br H Br H 3 CH₂C(CH₃)₂OH C═O Br H H Br 3 CH₂CHF₂ C═O HI H I 2 CH₂CF₃ C═O I H I H 2 CH₂CH(CH₃)₂ C═O I H H I 2 CH₂C(CH₃)₃ C═O HCH₃ H CH₃ 2 H₂C—C≡N C═O CH₃ H CH₃ H 2 H₂C—C≡CH C═O CH₃ H H CH₃ 2CH(CH₃)₂ C═O H C₂H₅ H C₂H₅ 2 CH₂CH₂OH C═O C₂H₅ H C₂H₅ H 2 CH₂CH(CH₃)OHC═O C₂H₅ H H C₂H₅ 2 CH(CH₃)CH₂OH C═O H i-C₃H₇ H i-C₃H₇ 2CH(CH₃)CH(CH₃)OH C═O i-C₃H₇ H i-C₃H₇ H 2 C(CH₃)₂CH₂OH C═O i-C₃H₇ H Hi-C₃H₇ 3 CH₂C(CH₃)₂OH C═S H Cl H Cl 3 CH₂CHF₂ C═S Cl H Cl H 3 CH₂CF₃ C═SCl H H Cl 3 CH₂CH(CH₃)₂ C═S H Br H Br 3 CH₂C(CH₃)₃ C═S Br H Br H 3H₂C—C≡N C═S Br H H Br 3 H₂C—C≡CH C═S H I H I 3 CH(CH₃)₂ C═S I H I H 3CH₂CH₂OH C═S I H H I 3 CH₂CH(CH₃)OH C═S H CH₃ H CH₃ 2 CH(CH₃)CH₂OH C═SCH₃ H CH₃ H 2 CH(CH₃)CH(CH₃)OH C═S CH₃ H H CH₃ 2 C(CH₃)₂CH₂OH C═S H C₂H₅H C₂H₅ 2 CH₂C(CH₃)₂OH C═S C₂H₅ H C₂H₅ H 2 CH₂CHF₂ C═S C₂H₅ H H C₂H₅ 2CH₂CF₃ C═S H i-C₃H₇ H i-C₃H₇ 2 CH₂CH(CH₃)₂ C═S i-C₃H₇ H i-C₃H₇ H 2CH₂C(CH₃)₃ C═S i-C₃H₇ H H i-C₃H₇ 3 H₂C—C≡N CH₂ H Cl H Cl 3 H₂C—C≡CH CH₂Cl H Cl H 3 CH(CH₃)₂ CH₂ Cl H H Cl 3 CH₂CH₂OH CH₂ H Br H Br 3CH₂CH(CH₃)OH CH₂ Br H Br H 3 CH(CH₃)CH₂OH CH₂ Br H H Br 2CH(CH₃)CH(CH₃)OH CH₂ H I H I 2 C(CH₃)₂CH₂OH CH₂ I H I H 2 CH₂C(CH₃)₂OHCH₂ I H H I 2 CH₂CHF₂ CH₂ H CH₃ H CH₃ 2 CH₂CF₃ CH₂ CH₃ H CH₃ H 2CH₂CH(CH₃)₂ CH₂ CH₃ H H CH₃ 3 CH₂C(CH₃)₃ CH₂ H C₂H₅ H C₂H₅ 3 H₂C—C≡N CH₂C₂H₅ H C₂H₅ H 3 H₂C—C≡CH CH₂ C₂H₅ H H C₂H₅ 3 CH(CH₃)₂ CH₂ H i-C₃H₇ Hi-C₃H₇ 3 CH₂CH₂OH CH₂ i-C₃H₇ H i-C₃H₇ H 3 CH₂CH(CH₃)OH CH₂ i-C₃H₇ H Hi-C₃H₇ 2 CH(CH₃)CH₂OH CH—OH H Cl H Cl 2 CH(CH₃)CH(CH₃)OH CH—OH Cl H Cl H2 C(CH₃)₂CH₂OH CH—OH Cl H H Cl 2 CH₂C(CH₃)₂OH CH—OH H Br H Br 2 CH₂CHF₂CH—OH Br H Br H 2 CH₂CF₃ CH—OH Br H H Br 3 CH₂CH(CH₃)₂ CH—OH H I H I 3CH₂C(CH₃)₃ CH—OH I H I H 3 H₂C—C≡N CH—OH I H H I 3 H₂C—C≡CH CH—OH H CH₃H CH₃ 3 CH(CH₃)₂ CH—OH CH₃ H CH₃ H 3 CH₂CH₂OH CH—OH CH₃ H H CH₃ 2CH₂CH(CH₃)OH CH—OH H C₂H₅ H C₂H₅ 2 CH(CH₃)CH₂OH CH—OH C₂H₅ H C₂H₅ H 2CH(CH₃)CH(CH₃)OH CH—OH C₂H₅ H H C₂H₅ 2 C(CH₃)₂CH₂OH CH—OH H i-C₃H₇ Hi-C₃H₇ 2 CH₂C(CH₃)₂OH CH—OH i-C₃H₇ H i-C₃H₇ H 2 CH₂CHF₂ CH—OH i-C₃H₇ H Hi-C₃H₇ 3 CH₂CF₃ CH—F H Cl H Cl 3 CH₂CH(CH₃)₂ CH—F Cl H Cl H 3 CH₂C(CH₃)₃CH—F Cl H H Cl 3 H₂C—C≡N CH—F H Br H Br 3 H₂C—C≡CH CH—F Br H Br H 3CH(CH₃)₂ CH—F Br H H Br 2 CH₂CH₂OH CH—F H I H I 2 CH₂CH(CH₃)OH CH—F I HI H 2 CH(CH₃)CH₂OH CH—F I H H I 2 CH(CH₃)CH(CH₃)OH CH—F H CH₃ H CH₃ 2C(CH₃)₂CH₂OH CH—F CH₃ H CH₃ H 2 CH₂C(CH₃)₂OH CH—F CH₃ H H CH₃ 3 CH₂CHF₂CH—F H C₂H₅ H C₂H₅ 3 CH₂CF₃ CH—F C₂H₅ H C₂H₅ H 3 CH₂CH(CH₃)₂ CH—F C₂H₅ HH C₂H₅ 3 CH₂C(CH₃)₃ CH—F H i-C₃H₇ H i-C₃H₇ 3 H₂C—C≡N CH—F i-C₃H₇ Hi-C₃H₇ H 3 H₂C—C≡CH CH—F i-C₃H₇ H H i-C₃H₇ 2 CH(CH₃)₂ S H Cl Cl Cl 2CH₂CH₂OH S Cl Cl H Cl 2 CH₂CH(CH₃)OH S H Br Br Br 2 CH(CH₃)CH₂OH S Br BrH Br 2 CH(CH₃)CH(CH₃)OH S H I I I 2 C(CH₃)₂CH₂OH S I I H I 3CH₂C(CH₃)₂OH S H CH₃ CH₃ CH₃ 3 CH₂CHF₂ S CH₃ CH₃ H CH₃ 3 CH₂CF₃ S H C₂H₅C₂H₅ C₂H₅ 3 CH₂CH(CH₃)₂ S C₂H₅ C₂H₅ H C₂H₅ 3 CH₂C(CH₃)₃ S H i-C₃H₇i-C₃H₇ i-C₃H₇ 3 H₂C—C≡N S i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 H₂C—C≡CH O H Cl Cl Cl2 CH(CH₃)₂ O Cl Cl H Cl 2 CH₂CH₂OH O H Br Br Br 2 CH₂CH(CH₃)OH O Br Br HBr 2 CH(CH₃)CH₂OH O H I I I 2 CH(CH₃)CH(CH₃)OH O I I H I 3 C(CH₃)₂CH₂OHO H CH₃ CH₃ CH₃ 3 CH₂C(CH₃)₂OH O CH₃ CH₃ H CH₃ 3 CH₂CHF₂ O H C₂H₅ C₂H₅C₂H₅ 3 CH₂CF₃ O C₂H₅ C₂H₅ H C₂H₅ 3 CH₂CH(CH₃)₂ O H i-C₃H₇ i-C₃H₇ i-C₃H₇3 CH₂C(CH₃)₃ O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 H₂C—C≡N S═O H Cl Cl Cl 2H₂C—C≡CH S═O Cl Cl H Cl 2 CH(CH₃)₂ S═O H Br Br Br 2 CH₂CH₂OH S═O Br Br HBr 2 CH₂CH(CH₃)OH S═O H I I I 2 CH(CH₃)CH₂OH S═O I I H I 3CH(CH₃)CH(CH₃)OH S═O H CH₃ CH₃ CH₃ 3 C(CH₃)₂CH₂OH S═O CH₃ CH₃ H CH₃ 3CH₂C(CH₃)₂OH S═O H C₂H₅ C₂H₅ C₂H₅ 3 CH₂CHF₂ S═O C₂H₅ C₂H₅ H C₂H₅ 3CH₂CF₃ S═O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH₂CH(CH₃)₂ S═O i-C₃H₇ i-C₃H₇ Hi-C₃H₇ 2 CH₂C(CH₃)₃ O═S═O H Cl Cl Cl 2 H₂C—C≡N O═S═O Cl Cl H Cl 2H₂C—C≡CH O═S═O H Br Br Br 2 CH(CH₃)₂ O═S═O Br Br H Br 2 CH₂CH₂OH O═S═O HI I I 2 CH₂CH(CH₃)OH O═S═O I I H I 3 CH(CH₃)CH₂OH O═S═O H CH₃ CH₃ CH₃ 3CH(CH₃)CH(CH₃)OH O═S═O CH₃ CH₃ H CH₃ 3 C(CH₃)₂CH₂OH O═S═O H C₂H₅ C₂H₅C₂H₅ 3 CH₂C(CH₃)₂OH O═S═O C₂H₅ C₂H₅ H C₂H₅ 3 CH₂CHF₂ O═S═O H i-C₃H₇i-C₃H₇ i-C₃H₇ 3 CH₂CF₃ O═S═O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 CH₂CH(CH₃)₂ NH HCl Cl Cl 2 CH₂C(CH₃)₃ NH Cl Cl H Cl 2 H₂C—C≡N NH H Br Br Br 2 H₂C—C≡CHNH Br Br H Br 2 CH(CH₃)₂ NH H I I I 2 CH₂CH₂OH NH I I H I 3 CH₂CH(CH₃)OHNH H CH₃ CH₃ CH₃ 3 CH(CH₃)CH₂OH NH CH₃ CH₃ H CH₃ 3 CH(CH₃)CH(CH₃)OH NH HC₂H₅ C₂H₅ C₂H₅ 3 C(CH₃)₂CH₂OH NH C₂H₅ C₂H₅ H C₂H₅ 3 CH₂C(CH₃)₂OH NH Hi-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH₂CHF₂ NH i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 CH₂CF₃ C═O HCl Cl Cl 2 CH₂CH(CH₃)₂ C═O Cl Cl H Cl 2 CH₂C(CH₃)₃ C═O H Br Br Br 2H₂C—C≡N C═O Br Br H Br 2 H₂C—C≡CH C═O H I I I 2 CH(CH₃)₂ C═O I I H I 3CH₂CH₂OH C═O H CH₃ CH₃ CH₃ 3 CH₂CH(CH₃)OH C═O CH₃ CH₃ H CH₃ 3CH(CH₃)CH₂OH C═O H C₂H₅ C₂H₅ C₂H₅ 3 CH(CH₃)CH(CH₃)OH C═O C₂H₅ C₂H₅ HC₂H₅ 3 C(CH₃)₂CH₂OH C═O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH₂C(CH₃)₂OH C═O i-C₃H₇i-C₃H₇ H i-C₃H₇ 2 CH₂CHF₂ C═S H Cl Cl Cl 2 CH₂CF₃ C═S Cl Cl H Cl 2CH₂CH(CH₃)₂ C═S H Br Br Br 2 CH₂C(CH₃)₃ C═S Br Br H Br 2 H₂C—C≡N C═S H II I 2 H₂C—C≡CH C═S I I H I 3 CH(CH₃)₂ C═S H CH₃ CH₃ CH₃ 3 CH₂CH₂OH C═SCH₃ CH₃ H CH₃ 3 CH₂CH(CH₃)OH C═S H C₂H₅ C₂H₅ C₂H₅ 3 CH(CH₃)CH₂OH C═SC₂H₅ C₂H₅ H C₂H₅ 3 CH(CH₃)CH(CH₃)OH C═S H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3C(CH₃)₂CH₂OH C═S i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 CH₂C(CH₃)₂OH CH₂ H Cl Cl Cl 2CH₂CHF₂ CH₂ Cl Cl H Cl 2 CH₂CF₃ CH₂ H Br Br Br 2 CH₂CH(CH₃)₂ CH₂ Br Br HBr 2 CH₂C(CH₃)₃ CH₂ H I I I 2 H₂C—C≡N CH₂ I I H I 3 H₂C—C≡CH CH₂ H CH₃CH₃ CH₃ 3 CH(CH₃)₂ CH₂ CH₃ CH₃ H CH₃ 3 CH₂CH₂OH CH₂ H C₂H₅ C₂H₅ C₂H₅ 3CH₂CH(CH₃)OH CH₂ C₂H₅ C₂H₅ H C₂H₅ 3 CH(CH₃)CH₂OH CH₂ H i-C₃H₇ i-C₃H₇i-C₃H₇ 3 CH(CH₃)CH(CH₃)OH CH₂ i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 C(CH₃)₂CH₂OHCH—OH H Cl Cl Cl 2 CH₂C(CH₃)₂OH CH—OH Cl Cl H Cl 2 CH₂CHF₂ CH—OH H Br BrBr 2 CH₂CF₃ CH—OH Br Br H Br 2 CH₂CH(CH₃)₂ CH—OH H I I I 2 CH₂C(CH₃)₃CH—OH I I H I 3 H₂C—C≡N CH—OH H CH₃ CH₃ CH₃ 3 H₂C—C≡CH CH—OH CH₃ CH₃ HCH₃ 3 CH(CH₃)₂ CH—OH H C₂H₅ C₂H₅ C₂H₅ 3 CH₂CH₂OH CH—OH C₂H₅ C₂H₅ H C₂H₅3 CH₂CH(CH₃)OH CH—OH H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH(CH₃)CH₂OH CH—OH i-C₃H₇i-C₃H₇ H i-C₃H₇ 2 CH(CH₃)CH(CH₃)OH CH—F H Cl Cl Cl 2 C(CH₃)₂CH₂OH CH—FCl Cl H Cl 2 CH₂C(CH₃)₂OH CH—F H Br Br Br 2 CH₂CHF₂ CH—F Br Br H Br 2CH₂CF₃ CH—F H I I I 2 CH₂CH(CH₃)₂ CH—F I I H I 3 CH₂C(CH₃)₃ CH—F H CH₃CH₃ CH₃ 3 H₂C—C≡N CH—F CH₃ CH₃ H CH₃ 3 H₂C—C≡CH CH—F H C₂H₅ C₂H₅ C₂H₅ 3CH(CH₃)₂ CH—F C₂H₅ C₂H₅ H C₂H₅ 3 CH₂CH₂OH CH—F H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3CH₂CH(CH₃)OH CH—F i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 CH(CH₃)CH₂OH S I H Cl H 2CH(CH₃)CH(CH₃)OH S═O I H H Cl 2 C(CH₃)₂CH₂OH O═S═O Br H Cl H 2CH₂C(CH₃)₂OH CH₂ Br H H Cl 2 CH₂CHF₂ C═O Br H I H 3 CH₂CF₃ C═S Br H H I3 CH₂CH(CH₃)₂ CH—OH I H Br H 3 CH₂C(CH₃)₃ CH—F I H H Br 3 H₂C—C≡N O I ClH Cl 3 H₂C—C≡CH NH Br Cl H Cl

TABLE 12

m Y X² X³ X⁴ X⁵ 2 S H Cl H H 2 S H H H Cl 2 S H H Cl H 2 S H Cl H Cl 2 SH Cl Cl Cl 2 O H Cl H H 2 O H H H Cl 2 O H H Cl H 2 O H Cl H Cl 2 O H ClCl Cl 2 CH₂ H Cl H H 2 CH₂ H H H Cl 2 CH₂ H H Cl H 2 CH₂ H Cl H Cl 2 CH₂H Cl Cl Cl 2 C═O H H H Cl 2 C═O H H Cl H 2 C═O H Cl H Cl 2 C═O H Cl ClCl 3 S H Cl H H 3 S H H H Cl 3 S H H Cl H 3 S H Cl H Cl 3 S H Cl Cl Cl 3O H Cl H H 3 O H H H Cl 3 O H H Cl H 3 O H Cl H Cl 3 O H Cl Cl Cl 3 CH₂H Cl H H 3 CH₂ H H H Cl 3 CH₂ H H Cl H 3 CH₂ H Cl H Cl 3 CH₂ H Cl Cl Cl3 C═O H H H Cl 3 C═O H H Cl H 3 C═O H Cl H Cl 3 C═O H Cl Cl Cl 2 S H CH₃H H 2 S H H H CH₃ 2 S H H CH₃ H 2 S H CH₃ H CH₃ 2 S H CH₃ CH₃ CH₃ 2 O HCH₃ H H 2 O H H H CH₃ 2 O H H CH₃ H 2 O H CH₃ H CH₃ 2 O H CH₃ CH₃ CH₃ 2CH₂ H CH₃ H H 2 CH₂ H H H CH₃ 2 CH₂ H H CH₃ H 2 CH₂ H CH₃ H CH₃ 2 CH₂ HCH₃ CH₃ CH₃ 2 C═O H H H CH₃ 2 C═O H H CH₃ H 2 C═O H CH₃ H CH₃ 2 C═O HCH₃ CH₃ CH₃ 3 S H CH₃ H H 3 S H H H CH₃ 3 S H H CH₃ H 3 S H CH₃ H CH₃ 3S H CH₃ CH₃ CH₃ 3 O H CH₃ H H 3 O H H H Cl 3 O H H CH₃ H 3 O H CH₃ H CH₃3 O H CH₃ CH₃ CH₃ 3 CH₂ H CH₃ H H 3 CH₂ H H H CH₃ 3 CH₂ H H CH₃ H 3 CH₂H CH₃ H CH₃ 3 CH₂ H CH₃ CH₃ CH₃ 3 C═O H H H CH₃ 3 C═O H H CH₃ H 3 C═O HCH₃ H CH₃ 3 C═O H CH₃ CH₃ CH₃ 3 S H Cl H Br 3 S H CH₂CH₃ CH₃ Br 3 S HOCH₃ H OCH₃ 2 S H Cl H H 2 S H H H Cl 2 S H H Cl H 2 S Cl H H H 2 S H BrH H 2 S H H H Br 3 S H H Br H 3 S Br H H H 3 S H I H H 3 S H H H I 3 S HH I H 3 S I H H H 4 S H CH₃ H H 4 S H H H CH₃ 4 S H H CH₃ H 4 S CH₃ H HH 4 S H C₂H₅ H H 4 S H H H C₂H₅ 5 S H H C₂H₅ H 5 S C₂H₅ H H H 5 S H H Hi-C₃H₇ 5 S H H H i-C₃H₇ 5 S H H i-C₃H₇ H 5 S i-C₃H₇ H H H 2 O H Cl H H 2O H H H Cl 2 O H H Cl H 2 O Cl H H H 2 O H Br H H 2 O H H H Br 3 O H HBr H 3 O Br H H H 3 O H I H H 3 O H H H I 3 O H H I H 3 O I H H H 4 O HCH₃ H H 4 O H H H CH₃ 4 O H H CH₃ H 4 O CH₃ H H H 4 O H C₂H₅ H H 4 O H HH C₂H₅ 5 O H H C₂H₅ H 5 O C₂H₅ H H H 5 O H i-C₃H₇ H H 5 O H H H i-C₃H₇ 5O H H i-C₃H₇ H 5 O i-C₃H₇ H H H 2 S═O H Cl H H 2 S═O H H H Cl 2 S═O H HCl H 2 S═O Cl H H H 2 S═O H Br H H 2 S═O H H H Br 3 S═O H H Br H 3 S═OBr H H H 3 S═O H I H H 3 S═O H H H I 3 S═O H H I H 3 S═O I H H H 4 S═O HCH₃ H H 4 S═O H H H CH₃ 4 S═O H H CH₃ H 4 S═O CH H H H 4 S═O H C₂H₅ H H4 S═O H H H C₂H₅ 5 S═O H H C₂H₅ H 5 S═O C₂H₅ H H H 5 S═O H i-C₃H₇ H H 5S═O H H H i-C₃H₇ 5 S═O H H i-C₃H₇ H 5 S═O i-C₃H₇ H H H 2 O═S═O H Cl H H2 O═S═O H H H Cl 2 O═S═O H H Cl H 2 O═S═O Cl H H H 2 O═S═O H Br H H 2O═S═O H H H Br 3 O═S═O H H Br H 3 O═S═O Br H H H 3 O═S═O H I H H 3 O═S═OH H H I 3 O═S═O H H I H 3 O═S═O I H H H 4 O═S═O H CH₃ H H 4 O═S═O H H HCH₃ 4 O═S═O H H CH₃ H 4 O═S═O CH₃ H H H 4 O═S═O H C₂H₅ H H 4 O═S═O H H HC₂H₅ 5 O═S═O H H C₂H₅ H 5 O═S═O C₂H₅ H H H 5 O═S═O H H H i-C₃H₇ 5 O═S═OH H H i-C₃H₇ 5 O═S═O H H i-C₃H₇ H 5 O═S═O i-C₃H₇ H H H 2 NH H Cl H H 2NH H H H Cl 2 NH H H Cl H 2 NH Cl H H H 2 NH H Br H H 2 NH H H H Br 3 NHH H Br H 3 NH Br H H H 3 NH H I H H 3 NH H H H I 3 NH H H I H 3 NH I H HH 4 NH H CH₃ H H 4 NH H H H CH₃ 4 NH H H CH₃ H 4 NH CH₃ H H H 4 NH HC₂H₅ H H 4 NH H H H C₂H₅ 5 NH H H C₂H₅ H 5 NH C₂H₅ H H H 5 NH H i-C₃H₇ HH 5 NH H H H i-C₃H₇ 5 NH H H i-C₃H₇ H 5 NH i-C₃H₇ H H H 2 C═O H Cl H H 2C═O H H H Cl 2 C═O H H Cl H 2 C═O Cl H H H 2 C═O H Br H H 2 C═O H H H Br3 C═O H H Br H 3 C═O Br H H H 3 C═O H I H H 3 C═O H H H I 3 C═O H H I H3 C═O I H H H 4 C═O H CH₃ H H 4 C═O H H H CH₃ 4 C═O H H CH₃ H 4 C═O CH HH H 4 C═O H C₂H₅ H H 4 C═O H H H C₂H₅ 5 C═O H H C₂H₅ H 5 C═O C₂H₅ H H H5 C═O H i-C₃H₇ H H 5 C═O H H H i-C₃H₇ 5 C═O H H i-C₃H₇ H 5 C═O i-C₃H₇ HH H 2 C═S H Cl H H 2 C═S H H H Cl 2 C═S H H Cl H 2 C═S Cl H H H 2 C═S HBr H H 2 C═S H H H Br 3 C═S H H Br H 3 C═S Br H H H 3 C═S H I H H 3 C═SH H H I 3 C═S H H I H 3 C═S I H H H 4 C═S H CH₃ H H 4 C═S H H H CH₃ 4C═S H H CH₃ H 4 C═S CH H H H 4 C═S H C₂H₅ H H 4 C═S H H H C₂H₅ 5 C═S H HC₂H₅ H 5 C═S C₂H₅ H H H 5 C═S H i-C₃H₇ H H 5 C═S H H H i-C₃H₇ 5 C═S H Hi-C₃H₇ H 5 C═S i-C₃H₇ H H H 2 CH₂ H Cl H H 2 CH₂ H H H Cl 2 CH₂ H H Cl H2 CH₂ Cl H H H 2 CH₂ H Br H H 2 CH₂ H H H Br 3 CH₂ H H Br H 3 CH₂ Br H HH 3 CH₂ H I H H 3 CH₂ H H H I 3 CH₂ H H I H 3 CH₂ I H H H 4 CH₂ H CH₃ HH 4 CH₂ H H H CH₃ 4 CH₂ H H CH₃ H 4 CH₂ CH₃ H H H 4 CH₂ H C₂H₅ H H 4 CH₂H H H C₂H₅ 5 CH₂ H H C₂H₅ H 5 CH₂ C₂H₅ H H H 5 CH₂ H i-C₃H₇ H H 5 CH₂ HH H i-C₃H₇ 5 CH₂ H H i-C₃H₇ H 5 CH₂ i-C₃H₇ H H H 2 CH—OH H Cl H H 2CH—OH H H H Cl 2 CH—OH H H Cl H 2 CH—OH Cl H H H 2 CH—OH H Br H H 2CH—OH H H H Br 3 CH—OH H H Br H 3 CH—OH Br H H H 3 CH—OH H I H H 3 CH—OHH H H I 3 CH—OH H H I H 3 CH—OH I H H H 4 CH—OH H CH₃ H H 4 CH—OH H H HCH₃ 4 CH—OH H H CH₃ H 4 CH—OH CH₃ H H H 4 CH—OH H C₂H₅ H H 4 CH—OH H H HC₂H₅ 5 CH—OH H H C₂H₅ H 5 CH—OH C₂H₅ H H H 5 CH—OH H i-C₃H₇ H H 5 CH—OHH H H i-C₃H₇ 5 CH—OH H H i-C₃H₇ H 5 CH—OH i-C₃H₇ H H H 2 CH—F H Cl H H 2CH—F H H H Cl 2 CH—F H H Cl H 2 CH—F Cl H H H 2 CH—F H Br H H 2 CH—F H HH Br 3 CH—F H H Br H 3 CH—F Br H H H 3 CH—F H I H H 3 CH—F H H H I 3CH—F H H I H 3 CH—F I H H H 4 CH—F H CH₃ H H 4 CH—F H H H CH₃ 4 CH—F H HCH₃ H 4 CH—F CH₃ H H H 4 CH—F H C₂H₅ H H 4 CH—F H H H C₂H₅ 5 CH—F H HC₂H₅ H 5 CH—F C₂H₅ H H H 5 CH—F H i-C₃H₇ H H 5 CH—F H H H i-C₃H₇ 5 CH—FH H i-C₃H₇ H 5 CH—F i-C₃H₇ H H H 2 S H Cl H Cl 2 S Cl H Cl H 2 S Cl H HCl 2 S H Br H Br 2 S Br H Br H 2 S Br H H Br 3 S H I H I 3 S I H I H 3 SI H H I 3 S H CH₃ H CH₃ 3 S CH₃ H CH₃ H 3 S CH₃ H H CH₃ 4 S H C₂H₅ HC₂H₅ 4 S C₂H₅ H C₂H₅ H 4 S C₂H₅ H H C₂H₅ 4 S H i-C₃H₇ H i-C₃H₇ 4 Si-C₃H₇ H i-C₃H₇ H 4 S i-C₃H₇ H H i-C₃H₇ 5 O H Cl H Cl 5 O Cl H Cl H 5 OCl H H Cl 5 O H Br H Br 5 O Br H Br H 5 O Br H H Br 2 O H I H I 2 O I HI H 2 O I H H I 2 O H CH₃ H CH₃ 2 O CH₃ H CH₃ H 2 O CH₃ H H CH₃ 3 O HC₂H₅ H C₂H₅ 3 O C₂H₅ H C₂H₅ H 3 O C₂H₅ H H C₂H₅ 3 O H i-C₃H₇ H i-C₃H₇ 3O i-C₃H₇ H i-C₃H₇ H 3 O i-C₃H₇ H H i-C₃H₇ 4 S═O H Cl H Cl 4 S═O Cl H ClH 4 S═O Cl H H Cl 4 S═O H Br H Br 4 S═O Br H Br H 4 S═O Br H H Br 5 S═OH I H I 5 S═O I H I H 5 S═O I H H I 5 S═O H CH₃ H CH₃ 5 S═O CH₃ H CH₃ H5 S═O CH H H CH₃ 2 S═O H C₂H₅ H C₂H₅ 2 S═O C₂H₅ H C₂H₅ H 2 S═O C₂H₅ H HC₂H₅ 2 S═O H i-C₃H₇ H i-C₃H₇ 2 S═O i-C₃H₇ H i-C₃H₇ H 2 S═O i-C₃H₇ H Hi-C₃H₇ 3 O═S═O H Cl H Cl 3 O═S═O Cl H Cl H 3 O═S═O Cl H H Cl 3 O═S═O HBr H Br 3 O═S═O Br H Br H 3 O═S═O Br H H Br 4 O═S═O H I H I 4 O═S═O I HI H 4 O═S═O I H H I 4 O═S═O H CH₃ H CH₃ 4 O═S═O CH₃ H CH₃ H 4 O═S═O CH₃H H CH₃ 5 O═S═O H C₂H₅ H C₂H₅ 5 O═S═O C₂H₅ H C₂H₅ H 5 O═S═O C₂H₅ H HC₂H₅ 5 O═S═O H i-C₃H₇ H i-C₃H₇ 5 O═S═O i-C₃H₇ H i-C₃H₇ H 5 O═S═O i-C₃H₇H H i-C₃H₇ 2 NH H Cl H Cl 2 NH Cl H Cl H 2 NH Cl H H Cl 2 NH H Br H Br 2NH Br H Br H 2 NH Br H H Br 2 NH H I H I 3 NH I H I H 3 NH I H H I 3 NHH CH₃ H CH₃ 3 NH CH₃ H CH₃ H 3 NH CH₃ H H CH₃ 3 NH H C₂H₅ H C₂H₅ 4 NHC₂H₅ H C₂H₅ H 4 NH C₂H₅ H H C₂H₅ 4 NH H i-C₃H₇ H i-C₃H₇ 4 NH i-C₃H₇ Hi-C₃H₇ H 4 NH i-C₃H₇ H H i-C₃H₇ 4 C═O H Cl H Cl 5 C═O Cl H Cl H 5 C═O ClH H Cl 5 C═O H Br H Br 5 C═O Br H Br H 5 C═O Br H H Br 2 C═O H I H I 2C═O I H I H 2 C═O I H H I 2 C═O H CH₃ H CH₃ 2 C═O CH₃ H CH₃ H 2 C═O CH₃H H CH₃ 3 C═O H C₂H₅ H C₂H₅ 3 C═O C₂H₅ H C₂H₅ H 3 C═O C₂H₅ H H C₂H₅ 3C═O H i-C₃H₇ H i-C₃H₇ 3 C═O i-C₃H₇ H i-C₃H₇ H 3 C═O i-C₃H₇ H H i-C₃H₇ 4C═S H Cl H Cl 4 C═S Cl H Cl H 4 C═S Cl H H Cl 4 C═S H Br H Br 4 C═S Br HBr H 4 C═S Br H H Br 5 C═S H I H I 5 C═S I H I H 5 C═S I H H I 5 C═S HCH₃ H CH₃ 5 C═S CH H CH₃ H 5 C═S CH H H CH₃ 2 C═S H C₂H₅ H C₂H₅ 2 C═SC₂H₅ H C₂H₅ H 2 C═S C₂H₅ H H C₂H₅ 2 C═S H i-C₃H₇ H i-C₃H₇ 2 C═S i-C₃H₇ Hi-C₃H₇ H 2 C═S i-C₃H₇ H H i-C₃H₇ 3 CH₂ H Cl H Cl 3 CH₂ Cl H Cl H 3 CH₂Cl H H Cl 3 CH₂ H Br H Br 3 CH₂ Br H Br H 3 CH₂ Br H H Br 4 CH₂ H I H I4 CH₂ I H I H 4 CH₂ I H H I 4 CH₂ H CH₃ H CH₃ 4 CH₂ CH₃ H CH₃ H 4 CH₂CH₃ H H CH₃ 5 CH₂ H C₂H₅ H C₂H₅ 5 CH₂ C₂H₅ H C₂H₅ H 5 CH₂ C₂H₅ H H C₂H₅5 CH₂ H i-C₃H₇ H i-C₃H₇ 5 CH₂ i-C₃H₇ H i-C₃H₇ H 5 CH₂ i-C₃H₇ H H i-C₃H₇2 CH—OH H Cl H Cl 2 CH—OH Cl H Cl H 2 CH—OH Cl H H Cl 2 CH—OH H Br H Br2 CH—OH Br H Br H 2 CH—OH Br H H Br 3 CH—OH H I H I 3 CH—OH I H I H 3CH—OH I H H I 3 CH—OH H CH₃ H CH₃ 3 CH—OH CH₃ H CH₃ H 3 CH—OH CH₃ H HCH₃ 4 CH—OH H C₂H₅ H C₂H₅ 4 CH—OH C₂H₅ H C₂H₅ H 4 CH—OH C₂H₅ H H C₂H₅ 4CH—OH H i-C₃H₇ H i-C₃H₇ 4 CH—OH i-C₃H₇ H i-C₃H₇ H 4 CH—OH i-C₃H₇ H Hi-C₃H₇ 5 CH—F H Cl H Cl 5 CH—F Cl H Cl H 5 CH—F Cl H H Cl 5 CH—F H Br HBr 5 CH—F Br H Br H 5 CH—F Br H H Br 2 CH—F H I H I 2 CH—F I H I H 2CH—F I H H I 2 CH—F H CH₃ H CH₃ 2 CH—F CH₃ H CH₃ H 2 CH—F CH₃ H H CH₃ 3CH—F H C₂H₅ H C₂H₅ 3 CH—F C₂H₅ H C₂H₅ H 3 CH—F C₂H₅ H H C₂H₅ 3 CH—F Hi-C₃H₇ H i-C₃H₇ 3 CH—F i-C₃H₇ H i-C₃H₇ H 3 CH—F i-C₃H₇ H H i-C₃H₇ 4 S HCl Cl Cl 4 S Cl Cl H Cl 4 S H Br Br Br 4 S Br Br H Br 4 S H I I I 4 S II H I 5 S H CH₃ CH₃ CH₃ 5 S CH₃ CH₃ H CH₃ 5 S H C₂H₅ C₂H₅ C₂H₅ 5 S C₂H₅C₂H₅ H C₂H₅ 5 S H i-C₃H₇ i-C₃H₇ i-C₃H₇ 5 S i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 O HCl Cl Cl 2 O Cl Cl H Cl 2 O H Br Br Br 2 O Br Br H Br 2 O H I I I 2 O II H I 3 O H CH₃ CH₃ CH₃ 3 O CH₃ CH₃ H CH₃ 3 O H C₂H₅ C₂H₅ C₂H₅ 3 O C₂H₅C₂H₅ H C₂H₅ 3 O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 4 S═OH Cl Cl Cl 4 S═O Cl Cl H Cl 4 S═O H Br Br Br 4 S═O Br Br H Br 4 S═O H II I 4 S═O I I H I 5 S═O H CH₃ CH₃ CH₃ 5 S═O CH CH₃ H CH₃ 5 S═O H C₂H₅C₂H₅ C₂H₅ 5 S═O C₂H₅ C₂H₅ H C₂H₅ 5 S═O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 5 S═Oi-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 O═S═O H Cl Cl Cl 2 O═S═O Cl Cl H Cl 2 O═S═O HBr Br Br 2 O═S═O Br Br H Br 2 O═S═O H I I I 2 O═S═O I I H I 3 O═S═O HCH₃ CH₃ CH₃ 3 O═S═O CH CH₃ H CH₃ 3 O═S═O H C₂H₅ C₂H₅ C₂H₅ 3 O═S═O C₂H₅C₂H₅ H C₂H₅ 3 O═S═O H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 O═S═O i-C₃H₇ i-C₃H₇ Hi-C₃H₇ 4 NH H Cl Cl Cl 4 NH Cl Cl H Cl 4 NH H Br Br Br 4 NH Br Br H Br 4NH H I I I 4 NH I I H I 5 NH H CH₃ CH₃ CH₃ 5 NH CH₃ CH₃ H CH₃ 5 NH HC₂H₅ C₂H₅ C₂H₅ 5 NH C₂H₅ C₂H₅ H C₂H₅ 5 NH H i-C₃H₇ i-C₃H₇ i-C₃H₇ 5 NHi-C₃H₇ i-C₃H₇ H i-C₃H₇ 2 C═O H Cl Cl Cl 2 C═O Cl Cl H Cl 2 C═O H Br BrBr 2 C═O Br Br H Br 2 C═O H I I I 2 C═O I I H I 3 C═O H CH₃ CH₃ CH₃ 3C═O CH₃ CH₃ H CH₃ 3 C═O H C₂H₅ C₂H₅ C₂H₅ 3 C═O C₂H₅ C₂H₅ H C₂H₅ 3 C═O Hi-C₃H₇ i-C₃H₇ i-C₃H₇ 3 C═O i-C₃H₇ i-C₃H₇ H i-C₃H₇ 4 C═S H Cl Cl Cl 4 C═SCl Cl H Cl 4 C═S H Br Br Br 4 C═S Br Br H Br 4 C═S H I I I 4 C═S I I H I5 C═S H CH₃ CH₃ CH₃ 5 C═S CH CH₃ H CH₃ 5 C═S H C₂H₅ C₂H₅ C₂H₅ 5 C═S C₂H₅C₂H₅ H C₂H₅ 5 C═S H i-C₃H₇ i-C₃H₇ i-C₃H₇ 5 C═S i-C₃H₇ i-C₃H₇ H i-C₃H₇ 2CH₂ H Cl Cl Cl 2 CH₂ Cl Cl H Cl 2 CH₂ H Br Br Br 2 CH₂ Br Br H Br 2 CH₂H I I I 2 CH₂ I I H I 3 CH₂ H CH₃ CH₃ CH₃ 3 CH₂ CH₃ CH₃ H CH₃ 3 CH₂ HC₂H₅ C₂H₅ C₂H₅ 3 CH₂ C₂H₅ C₂H₅ H C₂H₅ 3 CH₂ H i-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH₂i-C₃H₇ i-C₃H₇ H i-C₃H₇ 4 CH—OH H Cl Cl Cl 4 CH—OH Cl Cl H Cl 4 CH—OH HBr Br Br 4 CH—OH Br Br H Br 4 CH—OH H I I I 4 CH—OH I I H I 5 CH—OH HCH₃ CH₃ CH₃ 5 CH—OH CH₃ CH₃ H CH₃ 5 CH—OH H C₂H₅ C₂H₅ C₂H₅ 5 CH—OH C₂H₅C₂H₅ H C₂H₅ 5 CH—OH H i-C₃H₇ i-C₃H₇ i-C₃H₇ 5 CH—OH i-C₃H₇ i-C₃H₇ Hi-C₃H₇ 2 CH—F H Cl Cl Cl 2 CH—F Cl Cl H Cl 2 CH—F H Br Br Br 2 CH—F BrBr H Br 2 CH—F H I I I 2 CH—F I I H I 3 CH—F H CH₃ CH₃ CH₃ 3 CH—F CH₃CH₃ H CH₃ 3 CH—F H C₂H₅ C₂H₅ C₂H₅ 3 CH—F C₂H₅ C₂H₅ H C₂H₅ 3 CH—F Hi-C₃H₇ i-C₃H₇ i-C₃H₇ 3 CH—F i-C₃H₇ i-C₃H₇ H i-C₃H₇ 4 S I H Cl H 4 S═O IH H Cl 4 O═S═O Br H Cl H 4 CH₂ Br H H Cl 4 C═O Br H I H 4 C═S Br H H I 5CH—OH I H Br H 5 CH—F I H H Br 5 O I Cl H Cl 5 NH Br Cl H Cl

5.4 Grp94 Inhibitors of the Disclosure Exhibit Selective ParalogInhibition in Cells

Having established that the compounds of the disclosure selectivityinhibit Grp94, we next investigated the effect of Grp94 specificinhibitors in cells. As a test compound, we used the selective Grp94inhibitor PU-WS13, which has the following chemical structure:

We compared the in vitro effects of PU-WS13 with a selective Hsp90α/βinhibitor referred to as (PU-29F), which has the following chemicalstructure:

Selective target modulation of these compounds in cells was tested byseveral different readouts (FIG. 5). Specifically, we demonstrated thatPU-WS13 inhibited IGF-II secretion (FIG. 5a ) and Toll-like receptor 9(TLR9) trafficking (FIG. 5e ) in a dose-dependent manner. Both of theseare well-defined Grp94-mediated cellular events (Duerfeldt, A. S., etal. Development of a Grp94 inhibitor. J. Am. Chem. Soc. 134, 9796-9804(2012); Ostrovsky, O., Ahmed, N. T. & Argon, Y. The chaperone activityof GRP94 toward insulin-like growth factor II is necessary for thestress response to serum deprivation. Mol. Biol. Cell 20, 1855-1864(2009); At concentrations of PU-WS13 that inhibited Grp94 activity, weobserved no inhibition of Hsp90, as demonstrated by lack of Hsp70induction and AKT degradation (FIGS. 5 b, f, g), both of which arehallmarks of cytolosic Hsp90a inhibition. (Workman, P., Burrows, F.,Neckers, L. & Rosen, N. Drugging the cancer chaperone Hsp90:combinatorial therapeutic exploitation of oncogene addiction and tumorstress. Ann. N.Y. Acad. Sci. 1113, 202-216 (2007); Pearl, L. H.,Prodromou, C. & Workman, P. The Hsp90 molecular chaperone: an open andshut case for treatment. Biochem. J. 410, 439-453 (2008)) Conversely,treatment with the selective Hsp90α/β inhibitor PU-29F led to adose-dependent increase in Hsp90 levels and degradation of AKT (FIGS. 5b, f, g), while minimally effecting the Grp94 hallmarks (FIGS. 5 a, e,f). Importantly, PU-WS13 was not toxic to two non-malignant cell lines,C2C12 (mouse skeletal myoblasts) and HEK293 (human embryonic kidneycells) (FIGS. 5c, f ).

5.5 Therapeutic Uses of Grp94 Inhibitors of the Disclosure

The Grp94 inhibitors of the disclosure can be used to treat or preventany condition treatable or preventable by inhibiting the activity ofGrp94. Such conditions include, but are not limited to cancer,autoimmune diseases, neurodegenerative diseases and inflammatorydiseases. Due to their activity, the Grp94 inhibitors of the disclosureare advantageously useful in human medicine. When administered to ananimal, the Grp94 inhibitors of the disclosure can be administered as acomponent of a composition that comprises a pharmaceutically acceptablecarrier or excipient. The compositions of the disclosure can beadministered orally, intradermally, intramuscularly, intraperitoneally,parenterally, intravenously, subcutaneously, intranasally, epidurally,orally, sublingually, intracerebrally, intravaginally, transdermally,rectally or topically.

When a Grp94 inhibitor of the disclosure is incorporated for parenteraladministration by injection (e.g., continuous infusion or bolusinjection), the formulation for parenteral administration can be in theform of a suspension, solution, emulsion in an oily or aqueous vehicle,and such formulations can further comprise pharmaceutically necessaryadditives such as one or more stabilizing agents, suspending agents,dispersing agents, and the like. A Grp94 inhibitor of the disclosure canalso be in the form of a powder for reconstitution as an injectableformulation.

When a Grp94 inhibitor of the disclosure is formulated for oraladministration, the formulation can be in the in the form of tablets,capsules, gelcaps, caplets, lozenges, aqueous or oily solutions,suspensions, granules, powders, emulsions, or syrups. The oralformulation can include one or more pharmaceutically acceptableexcipients such as diluent, suspending agent, solubilizer, binder,disintegrant, preservative, coloring agent, lubricant. The Grp94inhibitors can be administered in a vesicle, and in particular, aliposome.

The Grp94 inhibitors of the disclosure are provided at doses that do notinhibit Hsp90π, Hsp90β and Trap-1. For instance, the Grp94 inhibitors ofthe disclosure can be administered at a dose in the range between 1mg/m² and 260 mg/m². In particular embodiments, the Grp94 inhibitors ofthe disclosure can be administered at a dose in the range between 2mg/m² and 100 mg/m². In other embodiments, the Grp94 inhibitors of thedisclosure can be administered at a dose in the range between 5 mg/m²and 50 mg/m². In still other embodiments, the Grp94 inhibitors of thedisclosure can be administered at a dose in the range between 5 mg/m²and 20 mg/m² or between 10 mg/m² and 20 mg/m².

5.5.1 Cancer

The Grp94 inhibitors of the disclosure can be used treat a variety ofcancers that are dependent on Hsp90 including but not limited tocolorectal cancer, pancreatic cancer, thyroid cancer, basal cellcarcinoma, melanoma, renal cell carcinoma, bladder cancer, prostatecancer, a lung cancer including small cell lung cancer and non-smallcell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancersincluding gastrointestinal stromal tumors, esophageal cancer, stomachcancer, liver cancer, gallbladder cancer, anal cancer, brain tumorsincluding gliomas, lymphomas including follicular lymphoma and diffuselarge B-cell lymphoma, leukemias, myelomas, myeloproliferative neoplasmsand gynecologic cancers including ovarian, cervical, and endometrialcancers.

The precise dose of the Grp94 inhibitor to be employed will depend on,e.g., the route of administration and the stage of the cancer. Inaccordance with the disclosure, the Grp94 inhibitors of the disclosurecan be administered to a patient such that the other Hsp90 paralogs arenot affected or affected to a minimal extent. Minimizing the inhibitionof the other Hsp90 paralogs can be achieved by an amount sufficient toinhibit binding of Grp94 to its client proteins without inhibitingbinding of the other Hsp90 paralogs. Accordingly, in one embodiment, aGrp94 inhibitor of the disclosure can be administered to a cancerpatient in an amount sufficient to inhibit binding of Grp94 to itsclient proteins without inhibition of the other HSP90 paralogs,including Hsp90π, HSP900 and Trap-1. As discussed herein, a particularadvantage of administering the Grp94 inhibitors of the disclosure atsuch a dosage range is that feed-back upregulation of antiapoptotic andresistance-mediating heat shock proteins (e.g., Hsp70) can besubstantially avoided. As such, the Grp94 inhibitors of the disclosurecan be administered to a patient without concomitant administration ofan Hsp70 inhibitor. Hence, in accordance with one aspect of thedisclosure, methods of treating cancer by treating a human patientsuffering from cancer without up-regulation of Hsp70 are provided. Suchmethods involve administration of a Grp94 inhibitor of the disclosure inan amount sufficient to inhibit binding of Grp94 to its client proteinswithout inhibiting binding of the other Hsp90 paralogs (i.e., Hsp90α,Hsp90β and/or Trap-1). In one embodiment, a Grp94 inhibitor of thedisclosure can be administered to a cancer patient in an amountsufficient to inhibit binding of Grp94 to its client proteins withoutinhibiting binding of client protein to other Hsp90 paralogs. In anotherembodiment, a Grp94 inhibitor of the disclosure can be administered to acancer patient in an amount sufficient to inhibit binding of Grp94 toits client proteins without up-regulation of Hsp70. Moreover, asdiscussed below, the Grp94 inhibitors of the disclosure are capable ofinducing apoptosis in cancer cells that express oncogenic proteins thatare dependent on Grp94 for survival and/or maintaining their function inthe survival or proliferation of cancer cells. For instance, asdiscussed below, Grp94 plays an important role in stabilizing particularreceptor tyrosine kinases (RTKs) at the plasma membrane, which allowsthe RTKs to be active in the development and progression of the tumors.The Grp94 inhibitors of the disclosure are capable of destabilizing themembrane RTKs, thereby inhibiting their signaling properties.

In certain embodiments, the Grp94 inhibitors of the disclosure can becombined with one or more other therapeutic agents for treating cancer.The therapeutic agents of the combination therapy may be administered atthe same time or may be administered sequentially. In particularembodiments, the Grp94 inhibitor can be administered with achemotherapeutic agent such as a toxin or a radioactive molecule. Inother embodiments, the Grp94 inhibitor can be administered together withan anti-angiogenic agent such as a VEGF antagonist. In yet otherembodiments, the Grp94 inhibitor can be administered together with aTNF-α antagonist. Specific examples of combination therapy will bediscussed below.

5.5.2 HER2 Dependent Tumors

With the Grp94 inhibitors of the disclosure, we investigated thespecific roles of Hsp90 paralogs on a classical Hsp90 client protein,HER2. HER2 is a receptor tyrosine kinase, which, when activated, leadsto the stimulation of many cancer-driving signaling pathways. Theexpression of HER2 is altered in many epithelial tumors such as breast,ovarian, gastric, and non-small-cell lung cancers, and HER2 levels havebeen shown to be inversely correlated with the prognosis of breastcancer. HER2 is also one of the most studied oncoprotein clients ofHsp90 and is one of the most sensitive to pan-Hsp90 inhibition.

The current view of the regulation of HER2 by Hsp90 chaperones comesfrom studies using pan-Hsp90 inhibitors. These suggest that the effectof these agents on HER2 is mediated by disrupting interactions betweenHsp90 and the HER2 cytoplasmic domain (Xu, W., Mimnaugh, E. G., Kim, J.S., Trepel, J. B. & Neckers, L. M. Hsp90, not Grp94, regulates theintracellular trafficking and stability of nascent ErbB2. Cell StressChaperones 7, 91-96 (2002)) leading to the poly-ubiquitination anddegradation of HER2 via the 26S proteasome. Pan-Hsp90 inhibitors alsoappear to act on Grp94 as it regulates the newly synthesized HER2 in theER, leading to HER2 instability and retention in the ER, with only traceubiquitination (Yarden, Y. & Sliwkowski, M. X. Untangling the ErbBsignaling network. Nat. Rev. Mol. Cell Biol. 2, 127-137 (2001).

The Grp94 inhibitors of the disclosure can be used to treat HER2dependent cancers such as breast cancer, ovarian cancer, gastric cancer,esophageal cancer and non-small-cell lung cancers. As discussed ingreater detail below, we have found that inhibition or depletion ofGrp94 in cells that overexpress HER2 results in apoptosis of the cellsalong with a mitigation or termination of the signaling event mediatedby HER2. Moreover, inhibition of Grp94 is not associated with feed-backupregulation of anti-apoptotic proteins, such as heat shock protein 70(Hsp70). As a result, the selective Grp94 inhibitors are capable ofinducing apoptosis of HER2 overexpressing cancer cells to a far greaterextent than pan-Hsp90 inhibitors, where upregulation of Hsp70 lessensthe anti-apoptotic effects of the inhibitor and may lead to resistance.Accordingly, the disclosure provides methods for selectively inducingapoptosis in HER2 overexpressing cancer cells. Moreover, the disclosureprovides methods of treating HER2 overexpressing cancers byadministering a therapeutically effective amount of a selective Grp94inhibitor.

In particular embodiments, the disclosure provides methods of treatingHER2 overexpressing breast cancers by administering a therapeuticallyeffective amount of a selective Grp94 inhibitor. In other embodiments,the disclosure provides methods of treating HER2 overexpressing ovariancancers by administering a therapeutically effective amount of aselective Grp94 inhibitor. In still other embodiments, the disclosureprovides methods of treating HER2 overexpressing gastric cancers byadministering a therapeutically effective amount of a selective Grp94inhibitor.

In some embodiments, the Grp94 inhibitors of the disclosure can be usedin combination with a therapeutic reagent that interferes with the HER2receptor (e.g., trastuzumab (herceptin)).

5.5.2.1 Hsp90 Paralogs Regulate HER2 in a Tumor-Specific Manner

To assess the role of the Hsp90 paralogs in HER2 dependent cancers, weused the Grp94 selective PU-WS13 and PU-H39 (FIG. 1), and the Hsp90α/βselective inhibitors PU-20F, PU-29F and PU-11, which have the followingchemical structures:

For comparison, we also employed the pan-Hsp90 inhibitor PU-H71 (FIG. 1a) to mimic, when relevant, the combined phenotypes observed withindividual selective Grp94 and Hsp90α/β inhibitors. In addition, weconfirmed relevant phenotypes by the use of at least threeparalog-specific siRNA constructs. We also performed confirmatoryparalog-selective affinity purifications with solid-support immobilizedprobes. For each compound, we controlled for selective target modulationin cells by several functional read-outs, including Hsp70 induction andRaf-1 and/or AKT degradation, as well as other cellular compartmentspecific effects as will be discussed below. Combined, these controlsprovide an independent measure of the cellular effects of selectiveparalog inhibition and allowed us to test the cellular effects of Grp94and Hsp90α/β inhibitors at concentrations that gave validated selectivetarget inhibition.

With this tool set in hand we probed two breast cancer cell lines, SKBr3(high HER2 expression) and MCF7 (low HER2 expression), for theindividual roles of Hsp90 paralogs in HER2 regulation. Surprisingly, wefound that the steady-state levels of HER2 were sensitive to selectiveinhibition of Grp94 in SKBr3 cells but not in MCF7 cells (FIG. 6a , topand FIG. 7a ). Knockdown of Grp94 levels by siRNA mimicked the effect ofthe Grp94 inhibitors. In both cases a similar reduction in thesteady-state levels of HER2 in SKBr3 cells, but not in MCF7 cells, wasobserved (FIG. 6b , top and FIG. 7b ).

Moreover, the Grp94 compounds of the disclosure fail to show substantialbinding and inhibition of major oncogenic kinases. For instance, bothPU-WS13 and PU-H39 were screened in the Discoverx scanEDGE. When testedat 10 uM, these compounds had no significant effect on any of the tested97 kinases. The tested kinases were distributed throughout the AGC,CAMK, CMGC, CK1, STE, TK, TKL, lipid, and atypical kinase families, plusimportant mutant forms. Furthermore, the effect of the Grp94 inhibitorycompounds is not directly on HER2 because lapatinib, a small moleculethat binds to the kinase domain of HER2, fails to mimic the phenotypeseen with PU-WS13 on SKBr3 cells. As seen, lapatinib does not disruptthe HER2 architecture at the plasma membrane. In contrast, our resultsclearly show that upon Grp94 inhibition both signaling and HER2-plasmastructures are disturbed. Collectively, these data link the biologicaleffects of the Grp94 inhibitors of the disclosure to their inhibition ofGrp94-mediated HER2 function.

In contrast, steady-state levels of HER2 were sensitive to Hsp90α/βinhibition (FIG. 6a and FIG. 7a ) and Hsp90α/β knockdown (FIG. 6b andFIG. 7b ) in both cell types. In high HER2 SKBr3 cells, HER2 levelsdecreased only at inhibitor concentrations that were indicative ofsimultaneous Hsp90a and Hsp90β inhibition (FIG. 6c ), mimicking the casefor another Hsp90α/β client protein, Raf-1 (Workman, P., Burrows, F.,Neckers, L. & Rosen, N. Drugging the cancer chaperone Hsp90:combinatorial therapeutic exploitation of oncogene addiction and tumorstress. Ann. N.Y. Acad. Sci. 1113, 202-216 (2007)) (FIG. 6c ). This wasconfirmed by siRNA knockdowns, where only dual Hsp90α/β siRNA knockdownmimicked the effect of Hsp90α/β inhibitors in this cell line (FIG. 6band FIG. 7b ). Selective siRNA knockdown of either Hsp90a or Hsp90β ledto only a partial reduction in HER2 levels (FIG. 6b ). In low HER2 MCF7cells however, HER2 levels decreased at lower inhibitor concentrationsthat were characteristic of selective binding to Hsp90α but not Hsp90β(FIG. 6a,c ). We also found a significant correlation in MCF7 cellsbetween HER2 degradation and Hsp90α- but not Hsp90β-, Grp94- andTrap-1-affinity (FIG. 6d , r²=0.83, 0.137, 0.217 and 0.005,respectively) (FIG. 6d , r²=0.83, 0.137, 0.217 and 0.005, respectively).HER2 also co-purified specifically with Hsp90α in these cells (FIG. 6e). Selective reduction of Hsp90α by means of siRNA, however, failed todecrease the level of HER2 in MCF7 cells (FIG. 6b and FIG. 7b ),possibly due to a feed-back induction of Hsp90β when Hsp90α issuppressed (FIG. 7 b.c).

Because HER2 is located in a membrane compartment associated with eitherthe ER and Golgi network or the plasma membrane, or is traffickedthrough the cytosol, we proceeded to investigate the effect of Hsp90specific inhibitors on HER2 in these locations. We found that in MCF7cells, the cytosolic HER2 protein levels as well as the activity ofother Hsp90-validated kinases, such as Raf-1 and ERK were rapidlyreduced by the Hsp90α/β inhibitor, but not by the Grp94 selectiveinhibitor (FIG. 6f ), further confirming that Hsp90 is the majorregulator of cytosolic HER2 in MCF7 cells.

To summarize, inhibition or downregulation of Grp94 leads to reducedsteady state levels of HER2 in high-HER2 SKBr3 cells, but not inlow-HER2 MCF7 cells. Similarly, inhibition or downregulation of bothHsp90a and Hsp90β reduces HER2 levels in high-HER2 SKBr3 cells, but notin low-HER2 MCF7 cells, where inhibition of the Hsp90a paralog alonesubstantially impairs HER2 stability. These data suggest atumor-specific involvement of the Hsp90 paralogs in the chaperoning ofHER2. Specifically, they propose the Hsp90a paralog to be sufficient forHER2 function in low-HER2 cells such as MCF7. On the other hand, incells with excessive amounts of HER2, such as SKBr3, all three Hsp90paralogs appear to play an important role.

5.5.2.2 Grp94 Regulates Plasma Membrane HER2 in SKBr3 Cells

We next investigated the unusual requirement for the involvement ofmultiple Hsp90 paralogs in regulating HER2 in SKBr3 cells. Unlike MCF7,SKBr3 cells express a high density of the HER2 protein at the plasmamembrane (Chavany, C. et al. p185erbB2 binds to GRP94 in vivo.Dissociation of the p185erbB2/GRP94 heterocomplex by benzoquinoneansamycins precedes depletion of p185erbB2. J. Biol. Chem. 271,4974-4977 (1996)), where interestingly we also detected Grp94 (FIG. 8a-d) but not Hsp90 (FIG. 8c,d ). Plasma membrane associated Grp94represents a small but substantial fraction of the total cellular Grp94(FIG. 8a-c ). We found that plasma membrane-associated Grp94co-localized (FIG. 8b , DMSO; FIG. 8c,d ) and co-precipitated with HER2(FIG. 8c,d ). Specific complex formation was confirmed both by chemicaland reciprocal immunopurification of Grp94/HER2 complexes (FIG. 8c,d )and by affinity purification performed with the Grp94 specific chemicaltool in cell lysates in which Grp94 levels were reduced byimmunopurification with Grp94 specific antibodies (FIG. 8e )

We next investigated the biological significance of the uniqueassociation of HER2 with Grp94 at the plasma membrane of SKBr3 cells.Because the Grp94 inhibitors described here target the ATP-bindingpocket of Grp94, they affect Grp94 chaperone activity. Therefore wehypothesized that Grp94 may act on HER2 at the plasma membrane tostabilize the protein and to regulate its function. Indeed, brieftreatment of SKBr3 cells with Grp94-selective compounds led to thedisruption of the circular architecture of HER2 at the plasma membrane,resulting in a “shredded” HER2 pattern (FIG. 8b,f PU-WS13). No sucheffect was observed upon direct HER2 inhibition with lapatinib (FIG. 8f, Lapatinib), a small molecule that binds to the ATP-regulatory pocketof HER2 (Kim, T. E. & Murren, J. R. Lapatinib ditosylateGlaxoSmithKline. IDrugs 6, 886-893 (2003)), further confirming that theeffect of PU-WS13 on HER2 was mediated through Grp94.

We found that upon Grp94 inhibition, HER2 molecules translocated toearly endosomes and plasma membrane-adjacent lysosomes (FIG. 8g , LAMP-1stain and FIG. 7d , EEA1 stain). Grp94-inhibited HER2 did notco-localize with ER and Golgi structures (FIG. 7d , Calnexin and 58k-9stains). The membrane but not the cytosolic HER2 molecules weresubstantially reduced in a time-dependent manner upon Grp94 inhibitionin SKBr3 cells (FIG. 8h ), altogether, further demonstrating that Grp94regulates HER2 specifically at the plasma membrane in SKBr3 cells.

In SKBr3 cells and other HER2-overexpressing breast cancer cells, thehigh-density HER2 tyrosine kinase formations at the cell membrane resultin increased signaling and activation of several survival andproliferation-inducing signaling pathways, such as those channeled byRaf-MAPK, AKT and STAT3 (Yarden, Y. & Sliwkowski, M. X. Untangling theErbB signaling network. Nat. Rev. Mol. Cell Biol. 2, 127-137 (2001)).For the case of the Raf-MAPK axis, HER2 promotes retention of Raf-1 inthe plasma membrane, resulting in prolonged activation of the MAP kinasecascade (Zhang, L., Bewick, M. & Lafrenie, R. M. EGFR and ErbB2differentially regulate Raf-1 translocation and activation. Lab. Invest.82, 71-78 (2002)). In further accord with a role for Grp94 in regulatingHER2 function at the plasma membrane, we found that pharmacologicinactivation of Grp94 in SKBr3 cells resulted in a rapid inhibition ofRaf-1-MAPK signaling at the membrane but not in the cytosol (FIG. 8i ).

Collectively, these findings indicate that in SKBr3 cells, Grp94chaperoning is needed to maintain a high-density HER2 architecture andan effective streamlining of its signaling at the plasma membrane butnot in the cytosol (FIG. 8j ). Without Grp94 chaperoning, the plasmamembrane architecture of HER2 becomes disrupted, leading to the shutdownof its signaling capacity. Altered HER2 molecules from plasma membranesbecome engulfed by lysosomes and endosomal structures, ultimatelyresulting in HER2 clearance.

5.5.2.3 Hsp90a p Regulate Cytosolic HER2 Species

Using our paralog-specific chemical toolset, we have shown that Grp94plays a key role in regulating HER2 at the plasma membrane of high-HER2SKBr3 cells. We next wanted to investigate the role of Hsp90 inregulating the cytosolic HER2. In agreement with the previously proposedspecialized role of Hsp90 on cytosolic HER2, Hsp90α/β inhibitors failedto disturb the membrane HER2 architecture in SKBr3 cells, and modifiedprimarily the cytosolic HER2 species (FIG. 8g ; PU-29F). As such, uponHsp90α/β inhibition, we observed a marked HER2 redistribution towardslysosomal and early endosomal structures that were distributedthroughout the cytosol (FIG. 8g , LAMP-1 stain and FIG. 7d , EEA1 stain;PU-29F). In addition, by 30 min following Hsp90α/β inhibition,steady-state levels of cytosolic but not membrane-associated HER2greatly decreased (FIG. 8h ), similar to what we have seen in MCF7 cells(FIG. 6f ). Following cytosolic HER2 depletion, we noted a decrease inplasma membrane associated HER2 (FIG. 8h ), confirming the previouslyproposed role of Hsp90 in the trafficking and regulation of thecytosolic HER2 species (Xu, W., Mimnaugh, E. G., Kim, J. S., Trepel, J.B. & Neckers, L. M. Hsp90, not Grp94, regulates the intracellulartrafficking and stability of nascent ErbB2. Cell Stress Chaperones 7,91-96 (2002)).

To summarize, our data point to distinct Hsp90 paralog requirements forHER2 regulation that are dictated by proteome alterations in the cell(FIG. 9). To chaperone the altered expression and activity of HER2 inHER2-overexpressing cells, where maintenance of ahigh-density/high-signaling HER2 species is a mechanism for itsoncogenic properties, the cell appears to utilize Hsp90α, Hsp90β andGrp94. Cytosolic HER2 requires both Hsp90α and Hsp90β. The aberrantlyhigh levels of plasma membrane HER2 require Grp94. In cells with lowHER2 expression, by contrast, the activity of Hsp90α alone appearssufficient to sustain HER2 function, although our knockdown studiesindicate that Hsp90β may compensate for the loss of Hsp90α expression inthese cells (FIG. 7b,c ).

5.5.2.4 Inhibition of Grp94 Alone is Sufficient to Reduce the Viabilityof HER2-Overexpressing Cells

Given the important role we identified for Grp94 in plasma membrane HER2stability and function in high-HER2 SKBr3 cells, we next asked whetherinactivating Grp94 reduced SKBr3 cancer cell viability. Indeed, bothGrp94 inhibition (FIG. 10a ) and Grp94 knockdown (FIG. 10b ) impairedSKBr3 viability. This effect was not limited to the SKBr3 cell line,since we observed that all other tested HER2-overexpressing breastcancer cells, such as AU565, BT474, MDA-MB-453 and MDA-MB-361, weresensitive to Grp94 inhibition (FIG. 10a ). Specifically, upon treatmentof these cells with PU-WS13, we noted a rapid accumulation of cells insub-G1 phase, observed as early as 1-2 h post-treatment (FIG. 10c ),cleavage of PARP (FIG. 10d,e and FIG. 1a ) and a substantial increase incells exhibiting markers of early- and late-stage apoptosis (FIG. 10f ).

Annexin V Apoptosis with 7-AAD has been specifically designed for theidentification of apoptotic and necrotic cells. Annexin V (or AnnexinA5) is a member of the annexin family of intracellular proteins thatbinds to phosphatidylserine (PS) in a calcium-dependent manner. PS isnormally only found on the intracellular leaflet of the plasma membranein healthy cells, but during early apoptosis, membrane asymmetry is lostand PS translocates to the external leaflet. Fluorochrome-labeledAnnexin V can then be used to specifically target and identify apoptoticcells. Annexin V binding alone cannot differentiate between apoptoticand necrotic cells. To help distinguish between the necrotic andapoptotic cells 7-amino-actinomycin D (7-AAD) is used. Early apoptoticcells will exclude 7-AAD, while late stage apoptotic cells will stainpositively, due to the passage of these dyes into the nucleus where theybind to DNA. 7-AAD (7-amino-actinomycin D) has a high DNA bindingconstant and is efficiently excluded by intact cells. It is useful forDNA analysis and dead cell discrimination during flow cytometricanalysis. When excited by 488 laser light, 7-AAD fluorescence isdetected in the far red range of the spectrum (650 nm long-pass filter).

As shown in FIG. 11 both the pan-Hsp90 inhibitor and the cytosolic Hsp90inhibitors failed to induce apoptosis in two HER2++ cells lines, SKBr3and AU565 as evidenced by no PARP cleavage (FIG. 11a,b ) and little tono apoptosis (FIG. 11d ).

Importantly, unlike the pan-Hsp90 and the cytosolic Hsp90 inhibitors,PU-WS13 failed to activate a feed-back heat shock response, as evidencedby little to no Hsp70 induction (FIG. 10d,e and FIG. 11). Hsp90α/βinhibition alone, despite substantially depleting HER2, was lesseffective at killing these cells and instead elicited a mostlycytostatic effect (FIG. 11b,c ). Neither inhibitor led to a substantialincrease in Grp78, the ER Hsp70 paralog, in these cells (FIG. 5b , FIG.11). Downregulation of Grp94 levels also failed to induce Grp78 in SKBr3cells (FIG. 7b ).

5.5.2.5 Grp94 Inhibitors can be Used to Treat HER2 OverexpressingGastric Cancers

Gastric cancer shows a poor prognosis and is the second leading cause ofcancer-related deaths. Its incidence is estimated at 934,000 cases, 56%of new cases are in Eastern Asia, 41% in China, and 11% in Japan.Although fluoropyrimidine- and platinum-based combination chemotherapyis the most widely accepted in the world at present, its benefit doesnot translate into higher overall survival rates. Despite recentadvances in the molecular understanding of gastric cancer, there is anoticeable lack of targeted therapies in clinical development for thismalignancy. Therefore, more effective therapies for gastric cancer arerequired. In gastric cancer, EGFR, HER2, and HER3 overexpression hasbeen identified and a relationship with prognosis is suggested.Therefore, inhibiting the signal transduction through heterodimersincluding HER2 possibly provides more benefit to patients with gastriccancer. Recently, the ToGA trial [a phase III study of trastuzumab(Herceptin) in HER2-positive advanced and inoperable gastric cancer]showed a survival benefit when trastuzumab was added to chemotherapy inHER2-overexpressing gastric cancer patients and the Food and DrugAdministration has approved trastuzumab for HER2-positive metastaticgastric and gastroesophageal junction cancer. Thus, anti-HER2 therapyhas been identified to be of clinical significance. Amplification ofHER2 has been associated with the intestinal pathologic subtype ofgastric cancer as well as with tumors arising from the gastroesophagealjunction. The largest analysis to date of the incidence of HER2amplification in gastric cancer was from the recently reported phase IIIclinical trial evaluating the combination of trastuzumab withchemotherapy in patients with metastatic gastric cancer. In this study,the overall rate of HER2 amplification was reported to be 22%, with ahigher percentage (34%) in patients with gastroesophageal junctiontumors.

We have found that gastric carcinomas expressing high levels of HER2 areparticularly sensitive to Grp94 inhibition. On the other hand, gastriccarcinomas that do not overexpress HER2 are not susceptible to Grp94inhibition therapy. The sensitivity of OE19, an esophagealadenocarcinoma with 100-fold amplification of the HER2 gene, to Grp94inhibition was tested using the Grp94 selective inhibitor PU-WS13.Likewise, the sensitivity of NCI-N87, a gastric carcinoma expressinghigh levels of HER2, was tested for sensitivity to Grp94 inhibition withthe Grp94 selective inhibitor PU-WS13. As shown in FIG. 12a , both theOE19 and NCI-N87 cells were highly susceptible to Grp94 inhibition. Onthe other hand, MKN74, a gastric adenocarcinoma with no HER2amplification was not sensitive to Grp94 inhibition. Moreover, as shownin FIG. 12b , there was a substantial increase in cells exhibitingmarkers of early- and late-stage apoptosis observed for the OE19 andNCI-N87 cells but not for the MKN74 cells.

5.5.3 EGFR Dependent Tumors

The epidermal growth factor receptor (EGFR) gene, located on chromosome7p12, encodes a 170 kDa membrane glycoprotein. Upon activation byspecific ligands such as EGF, its intrinsic kinase is activated andinitiates a number of signaling pathways. Upregulated EGFR signaling hasbeen correlated in a wide variety of tumors with progression to invasionand metastasis. EGFR was purified initially from the human squamous cellcarcinoma cell line A431, which overexpresses EGFR from 2- to 100-fold,resulting from a commensurate 3- to 110-fold increase in EGFR gene copynumber. Since then, many types of epithelial malignancies have beenshown to express increased levels of EGFR expression on the cellmembrane, with or without gene amplification. EGFR has been identifiedas a strong prognostic indicator in head and neck, breast, ovarian,cervical, bladder, and esophageal cancers. High EGFR expression has beenshown to correlate with poor survival in a range of tumors includingnasopharyngeal, NSCLC, ovarian, and breast. In patients withnasopharyngeal carcinoma, a significant correlation between high levelsof EGFR and poor survival has also been noted. In ovarian cancerspecimens, 61% scored positive for EGFR, and a significant correlationwas observed between EGFR expression and shorter overall andprogression-free survival. This study also correlated EGFR status withresistance to platinum-containing chemotherapy. In addition, severalstudies have reported that EGFR expression predicts for a significantlyshorter disease-free and overall survival in patients with breastcancer. Potentially explaining the association with poor patientoutcome, the expression of EGFR has been linked with resistance to bothhormonal therapies and chemotherapeutic agents. There is increasingevidence demonstrating that growth factor pathways are highlyinteractive with estrogen receptor signaling in the control of breastcancer growth. In tamoxifen-resistant breast cancer cell lines,antiestrogenic resistance is associated with upregulation of the EGFRpathway.

The Grp94 inhibitors of the disclosure can be used to treat EGFRdependent cancers such as pancreatic cancer, neck cancer, breast cancer,ovarian cancer, cervical cancer, bladder and esophageal cancers. We havefound that inhibition or depletion of Grp94 in cells that overexpressEGFR results in apoptosis of the cells along with a mitigation ortermination of the signaling event mediated by EGFR. Moreover,inhibition of Grp94 is not associated with feed-back upregulation ofanti-apoptotic proteins, including heat shock protein 70 (Hsp70). As aresult, the selective EGFR inhibitors are capable of inducing apoptosisof HER2 overexpressing cancer cells to a far greater extent thanpan-Hsp90 inhibitors, where upregulation of Hsp70 mitigates theanti-apoptotic effects of the inhibitor. Accordingly, the disclosureprovides methods for selectively inducing apoptosis in EGFRoverexpressing cancer cells. Moreover, the disclosure provides methodsof treating EGFR overexpressing cancers by administering atherapeutically effective amount of a selective Grp94 inhibitor.

In particular embodiments, the disclosure provides methods of treatingEGFR overexpressing breast cancers by administering a therapeuticallyeffective amount of a selective Grp94 inhibitor. In some suchembodiments, the breast cancer is triple negative breast cancer. Inother embodiments, the disclosure provides methods of treating EGFRoverexpressing pancreatic cancers by administering a therapeuticallyeffective amount of a selective Grp94 inhibitor. In still otherembodiments, the disclosure provides methods of treating HER2overexpressing ovarian cancers by administering a therapeuticallyeffective amount of a selective Grp94 inhibitor.

In some embodiments, the Grp94 inhibitors of the disclosure can be usedto treat endocrine-resistant breast and ovarian cancers (e.g., tumorsresistant to tamoxifen). The Grp94 inhibitors of the disclosure may beused in combination with am antiestrogen such as a selective estrogenreceptor modulator (e.g., tamoxifen) or an aromatase inhibitor (e.g.,exemestone or anastrozole).

The Grp94 inhibitors of the disclosure can be used to treat patientswith EGFR overexpressing triple negative breast cancer. As shown inFIGS. 13a-c , EGFR overexpressing triple negative breast cancer cellsare sensitive to the selective Grp94 inhibitor PU-WS13. The sensitivityof the EGFR overexpressing triple negative breast cancer cells wastested for the presence of apoptotic cells by Annexin V staining (13a,13b) and by immunoblotting for the presence of cleaved PARP (13c). Therewas a substantial increase in the triple negative breast cancer cellsexhibiting markers of early- and late-stage apoptosis (FIG. 13a,b ) andan increase in PARP cleavage following Grp94 but not Hsp90 inhibition(FIG. 13c ) Hence, Grp94 inhibition resulted in apoptosis of the triplenegative breast cancer cells. The Grp94 inhibitors of the disclosure canbe used to treat patients with EGFR overexpressing pancreatic cancer.Ligand activation of EGFR-family proteins (EGFR is a member of thereceptor tyrosine kinase superfamily of transmembrane proteins) resultsin perturbation of a variety of downstream signaling cascades. Based onstudies described herein, we have uncovered that Grp94 maintains thearchitecture of high density EGFR formations at the plasma membrane,particularly in cells where EGFR is required to channel the amplifiedsignaling through the receptor (e.g., EGFR overexpressing pancreaticcells). Hence, Grp94 inhibition results in a significant attenuation ofEGFR signaling.

As shown in FIG. 14, EGFR overexpressing cancer cells are sensitive tothe Grp94 inhibitor PU-WS13. EGFR levels are 10-17-fold higher in thePANC-1 cells relative to that observed in the Capan-2 cells. CFPAC alsoexpress low EGFR levels. HER2 levels are similar among the cell lines.The selective Grp94 inhibitor PU-WS13 effectively inhibited the growthof the EGFR overexpressing PANC-1 cells but had no effect on the Capan-2cells (FIG. 14a ). The Grp94 selective inhibitor had a modest effect onthe growth of the CFPAC cells (FIG. 14a ). Moreover, as shown in FIG.14b , there was a substantial increase in cells exhibiting markers ofearly- and late-stage apoptosis observed for the PANC-1 cells but notfor the Capan-2 cells. In contrast, the pan-Hsp90 inhibitor PU-H71 (FIG.1a ) and the HSP90α inhibitor PU29F had very little effect on inducingapoptosis of the PANC-1 cells (FIG. 15).

Of note, PANC1 EGFR-overexpressing cells are reported to be resistant tothe EGFR inhibitor erlotinib (Mol Cancer Ther 2006; 5:2051-2059)suggesting that inhibition of EGFR signaling by Grp94 inhibitors may bemore efficacious in pancreatic cancer than inhibition of EGFR by EGFRkinase inhibitors. Erlotinib (Tarceva, OSI-774, OSI Pharmaceuticals,Inc.) is a low molecular weight, orally bioavailable inhibitor of EGFRand exhibits >100-fold selectivity for EGFR over other receptor tyrosinekinases, including PDGFR, insulin-like growth factor-I receptor, andHER-2.

The Grp94 inhibitors of the disclosure can be used to treat EGFRdependent cancers that are resistant to therapy with EGFR inhibitors. Inone such embodiment, the cancer is pancreatic cancer that is resistantto therapy with EGFR inhibitors. The Grp94 inhibitor can be used incombination with an EGFR inhibitor. In particular embodiments, a Grp94inhibitor is used in combination with the EGFR inhibitor erlotinib inthe treatment of pancreatic cancer.

Aberrant epidermal growth factor receptor (EGFR) expression is detectedin up to 60% of ovarian cancers and occurs in all histologic subtypes.Further, aberrant EGFR expression is associated with poor outcome ofovarian cancer patients. Overexpression of the EGFR protein has beendetected in 9%-62% of human ovarian cancers; the differences infrequencies from these studies likely reflect utilization of differentantibodies and cutoffs for overexpression. EGFR gene amplification orprotein overexpression occurs across all epithelial ovarian cancerhistotypes. Increased EGFR expression has been associated with hightumor grade, high cell proliferation index, aberrant P53 expression, andpoor patient outcome (Siwak et al Journal of Oncology 2010; doi:10.1155/2010/568938). The Grp94 inhibitors of the disclosure can be usedto treat EGFR dependent ovarian cancers.

5.5.4 the Grp94 Inhibitor does not Affect RTK Expression and Activity inNormal Cells

FIG. 24 shows the activity of PU-WS13 in a panel of cancer cell linesdriven by overexpression of either HER2 or EGFR receptor tyrosinekinases. For comparison, the agent was also tested in a normal cellline, human mammary epithelial cells (HMECs). Note that the Grp94inhibitor PU-WS13 does not affect EGFR and its downstream signaling innormal cells characterized by normal expression and function of EGFR,such as in the HMEC cells. Without wishing to be bound by any particulartheory, it is believed that a Grp94 selective inhibitor may thereforehave a better therapeutic index than a direct RTK modulator (i.e. TKI orantibody) because it will act on the RTK only in conditions of oncogenicoverexpression (see EGFR in TNBC cell lines vs HMEC). Hence it should bedevoid of the side effects commonly associated with therapies directlyinhibiting RTKs (cardiac toxicity for trastuzumab and lapatinib,diarrhea, asthenia, and stomatitis for Canertinib, an irreversiblepan-HER TKI; diarrhea and rash for EGFR/HER2 TKIs due to RTK inhibitionin normal tissues). Grp94 inhibition should also be more active in EGFR+tumors than direct TKI. Approximately half of cases of triple-negativebreast cancer (TNBC) and inflammatory breast cancer (IBC) overexpressEGFR, nonetheless clinical trials testing EGFR inhibitors reported lackof or limited benefit (Masuda H. et al. Breast Cancer Res Treat. 2012November; 136(2):331-45). Without wishing to be bound by any particulartheory, it is believed that such ineffectiveness is due to a crosstalkbetween EGFR and c-Met or other RTKs, because strategies that knockeddown EGFR either by siRNA or by mixtures of antibodies that inducesrobust degradation of EGFR, led to reduced viability of TNBC cells(Mueller et al., Breast Cancer Res. 2012 Jul. 12; 14(4):R104; Ferraro etal., Proc Natl Acad Sci USA. 2013 Jan. 29; 110(5):1815-20.PMID:23319610). As per our findings, Grp94 inhibition also inducesrobust EGFR degradation and apoptosis in TNBC cells, and this effect mayprovide a therapeutic advantage over TKIs. This indicates that incertain tumors, such as in those addicted on survival on plasmaRTK-overexpression, Grp94 inhibition may provide better tumorsuppression than a pan-HSP90 inhibitor. While Grp94 inhibitiondownregulates RTK levels and their downstream signaling, similarly topan-HSP90 inhibitors, it fails to upregulate a feed-back stress response(i.e. Hsp70 induction).

Cell Lines

The cells, SKBr3, BT474, MDA-MB-468, HCC1806 and MDA-MB-453, wereobtained from the American Type Culture Collection (ATCC). Cells werecultured routinely in McCoy's 5A (10% FBS, SKBr3), DME/F12 (10% FBS,BT474 and MDA-MB-468), RPMI (10% FBS, HCC1806) and L-15 (20% FBS,MDA-MB-453) supplemented with 1% Glutamax and 1% penicillin andstreptomycin (Pen/Strep). HMEC cells were purchased from Lonza andcultured using Clonetics MEGM Bulletkit. When cultured, cells in L-15medium were kept in a humidified atmosphere without CO₂ at 37° C. andall other cell lines were incubated in the humidified cell incubatorswith CO₂ at 37° C.

Growth Inhibition Assay

We evaluated the antiproliferative effects of inhibitors using the dyeAlamar blue. This reagent offers a rapid objective measure of cellviability in cell culture, and it uses the indicator dye resazurin tomeasure the metabolic capacity of cells, an indicator of cell viability.Briefly, MDA-MB-468 cells were plated on Costar 96-well plates at 1500cells/well. Cells were allowed to incubate for 24 h at 37° C. beforedrug treatment. Drugs were added in triplicate at the indicatedconcentrations, and the plate was incubated for 72 h. Alamar Blue (440μM) was added, and the plate read 6 h later using Softmax Pro 6 software(Fluorescence intensity mode, excitation 530 nm, emission 580 nm, with560 nm dichroic mirror). Results were analyzed using GraphPad Prism 5.The percentage cell growth inhibition was calculated by comparingfluorescence readings obtained from treated versus control cells,accounting for initial cell population (time zero). The IC₅₀ wascalculated as the drug concentration that inhibits cell growth by 50%.

5.5.5 the Grp94 Inhibitors have a Higher Activity Against Tumor-RelatedGrp94 Function Versus Housekeeping (i.e. Normal, Physiological)Functions

FIG. 25 shows the activity of the Grp94 inhibitor PU-WS13 againsthousekeeping and tumor-related Grp94 functions. For this purpose we usedmice bearing MDA-MB-468 tumors (a triple-negative breast tumor withEGFR-overexpression). Because of the specific affinity of the providedGrp94 inhibitors for tumor Grp94, we performed PK/PD studies that aretailored for this purpose. In this PK/PD study we incorporated two timepoints, a 2 h and a 24 h time of sacrifice post-administration. Theearly time point is incorporated to test the biodistribution of theagent to the site of its action, the tumor; PU-WS13 was readilydistributed to tumor mass with ˜850 μM noted in tumor at 2 h versus 100μM in the plasma (FIG. 25A). At 24 h, the ratio of agent in tumor vsplasma increased to 200:1 from 7:1 at 2 h post administration of asingle dose, and the AUC^(0-24 h) tumor/plasma was 9134/1255, indicatingspecific retention of PU-WS13 in the tumor mass (FIG. 25A; AUC units areμM×h⁻¹). The concentration of PU-WS13 in the tumor at 24 hpost-administration of single dose of 75 mg/kg was 0.5 μM. Theassociated PD effect correlated with the tumor PK, i.e. was reflectiveof the tumor concentration of PU-WS13 and indicated partial suppressionof downstream EGFR signaling (FIG. 25B, see p-AKT and p-ERK inhibition),demonstrating that PU-WS13 engaged tumor Grp94 at this concentration. Wealso analyzed the potential suppression by PU-WS13 of normal Grp94functions. A “housekeeping” function for Grp94 was identified using aconditional knock-out mouse model; that study found a role for Grp94 innormal GI cells, i.e. regulation of the Wnt receptor LRP6 (Liu et al.,Proc Natl Acad Sci USA. 2013 Apr. 23; 110(17):6877-82). Because mostsmall molecules such as PU-WS13 are largely cleared via the GI track,the GI is the normal organ most exposed to agent over the time it spendsin the body. The AUC^(0-24 h) for stomach and the large intestine wasindeed 1.4 and 2-fold, respectively, higher than the tumor AUC^(0-24 h)(FIG. 25C); nonetheless, we could not detect a significant decrease inLRP6 (the Wnt receptor regulated in the normal GI by “housekeeping”Grp94) (FIG. 25D). We increased the administered dose of PU-WS13 to 150mg/kg; the AUC⁰⁻²⁴ h for the GI track increased by ˜6-fold, nonethelesswe observed no acute tox or change in LRP6 levels.

Four- to 6-week-old nu/nu athymic female mice were obtained from TaconicFarms. Experiments were carried out under an Institutional Animal Careand Use Committee approved protocol, and institutional guidelines forthe proper and humane use of animals in research were followed.MDA-MB-468 (1×10⁷ cells) were subcutaneously implanted in the rightflank of mice using a 20-gauge needle and allowed to grow. All micereceived Augmentin (amoxicillin/clavulanate potassium; SmithKlineBeecham) in their drinking water while on therapy.

For pharmacodynamic and pharmacokinetic assays, mice with establishedMDA-MB-468 tumors were given assigned doses of inhibitors or vehicle(intraperitoneally). Mice were euthanized by CO₂ asphyxiation and allrelevant tissues were harvested at a designated time after inhibitoradministration (formulated in 30% captisol in 60 mM citrate buffer).Tissues were flash frozen in liquid nitrogen, and divided into twohalves. One-half of the frozen tissues were dried and weighed prior tohomogenization in 750 μl water/acetonitrile (70:30) solution. Sampleswere extracted with 600 μl of methylene chloride twice from tissues andthen dried in the genevac. Later, samples were dissolved in solvent (75%water: 25% acetonitrile+0.1% formic acid), spun down at 4° C. andconcentrations of the inhibitors in tissue were determined byhigh-performance LC-MS/MS using haloperidol as the internal standard.Compound analysis was performed on the 6410 LC-MS/MS system (AgilentTechnologies). A Zorbax Eclipse XDB-C18 column (2.1×50 mm, 3.5 μm) wasused for the LC separation, and the analyte was eluted under anisocratic condition (65% H₂O+0.1% HCOOH: 35% CH₃CN) for 5 minutes at aflow rate of 0.35 ml/min.

The other half of tumor tissues were evaluated for changes in EGFR andother PD markers as established in our laboratory. Briefly, tumortissues were mixed with steel beads and tissue extraction buffer (50 mMTris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.25% sodium deoxycholate,0.5% NP40, 0.25% Triton X-100, protease inhibitors). The samples werehomogenized by the Bullet Blender (Next Advance, Inc) at 4° C. Thelysates were then transferred to a clean tube and centrifuge at 13,200rpm for 5 min at 4° C. After quantifying the protein concentrations byBCA, 25-100 ug proteins were loaded into SDS-PAGE and subjected toimmunoblotting.

Immunoblotting

Cells were either treated with DMSO (vehicle) or indicated compounds for24 hr and lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl,0.5% sodium deoxycholate and 0.5% NP40) supplemented with cocktailprotease inhibitors (Roche) to produce whole cell lysates. Proteinconcentrations were determined using BCA kit (Pierce) according to themanufacturer's instructions. The protein lysates (10-50 μg) wereelectrophoretically resolved by SDS-PAGE, transferred ontonitrocellulose membranes and probed with the indicated primaryantibodies against: HER2 (Zymed, 28004), EGFR (Cell Signaling, 4267),β-actin (Sigma, A1978), phospho-STAT3 (Cell Signaling, 9145), STAT3(Cell Signaling, 12640), Hsp70 (Stressgen, SPA-810), ERK1/2 (CellSignaling, 4695), phospho-ERK1/2 (Cell Signaling, 4370), phospho-AKT(Cell Signaling, 4060), AKT (Cell Signaling, 9272), cleaved PARP(Promega, G7341) and LRP6 (Cell Signaling, 2560). After washing off theexcess antibodies, the membranes were incubated with the correspondinghorseradish peroxidase (HRP) conjugated secondary antibody. Blots werevisualized by autoradiography using the Enhanced ChemiluminescenceDetection System (GE Healthcare) according to manufacturer'sinstructions. For all gels β-actin was used as a protein loadingcontrol.

5.5.6 IGF1R Dependent Tumors

The Grp94 inhibitors of the disclosure can be used to treat Insulingrowth factor 1 receptor (IGF1R) dependent tumors. In particular, theGrp94 inhibitors of the disclosure can be used in treating cancers withaltered expression of the IGFIR where the receptor is necessary forpathogenesis and tumor progression.

In addition to playing an important role in normal cell growth,maintenance and development, insulin-like growth factor receptor (IGF1R)and its ligands are also important in the establishment and maintenanceof the malignant phenotype. Binding of IGF-1 and IGF-II ligands to theIGF1R initiates a cascade of events leading to activation of mitogenicsignaling pathway (Ras/Raf/MAPK) and antiapoptotic/survival pathway(PI3K-Akt/mTOP), resulting in proliferation, transformation and survivalin tumor cells (D. LeRoith, et al., Cancer Lett., 195(2): 127-37 (2003),R. Baserga, et al., Int. J. Cancer; 107:873-7 (2003)). IGF1Roverexpression and/or enhanced activity have been observed in diversetumor types suggesting that the potential therapeutic use of agentstargeting this pathway is broad. IGF1R provides a critical survivalsignal in multiple tumor types. The expression of this receptor is anindicator of poor prognosis, thus, it has emerged as an attractive andcompelling target for cancer therapy to inhibit the progression ofmultiple tumor types in cancer patients. Various drug discoveryapproaches have been explored in recent years to modulate the functionof IGF1R. Approaches aimed at the reduction of receptor number orenzymatic activity using a variety of strategies in preclinical modelshave been shown to reverse the malignant phenotype in tumor cells. Thesestrategies include antisense (L. Long, et al., Cancer Res, 55(5): 1006-9(1995), D. Andrews et al., J. Clin. Oncol., 19(8):2189-200 (2001)),monoclonal antibody (C. Arteaga, et al., Cancer Res., 49(22):6237-41(1989)), small molecule inhibitors (M. Wittman, et al., J. Med. Chem.,Sep. 8; 48(18):5639-43 (2005), C. Garcia-Echeverria, et al., CancerCell, 5(3):231-9 (2004)), IGF-1 mimetic peptides (Z. Pietrzkowski, etal., Cancer Res., 53(5): 1102-6 (1993)) as well as dominant negativemutants that lack enzyme activity (C. D'Ambrosio, et al., Cancer Res,56(17): 4013-20 (1996)).

The disclosure provides evidence that Grp94 inhibitors are efficaciousin treating cancer with altered expression of the IGFIR and where thereceptor is necessary for pathogenesis and tumor progression. Inparticular, Grp94 inhibitors of the disclosure are capable of inducingapoptosis in IGFIR overexpressing cells. For instance, FIG. 16 showsthat the Grp94 selective inhibitor PU-WS13 is capable of inducingapoptosis in two IGFIR overexpressing Ewing sarcoma cell lines (A673 andTC71). Specifically, there was a substantial increase in the Ewingsarcoma cells exhibiting markers of early- and late-stage apoptosis.

5.5.7 TGFbeta Dependent Tumors

Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine thatregulates cell proliferation, apoptosis, differentiation, migration andinvasion. TGF-β signals through transmembrane type I (TβRI) and type II(TβRII) receptors to initiate downstream signaling. In the canonicalpathway, TGF-β binding to TβRII recruits and phosphorylates TβRI, whichresults in TβRI activation. Activated TβRI phosphorylates thereceptor-regulated Smad proteins Smnad2 and Smnad3. Phosphorylated Smad2and Smad3 then co-associate with Smad4, translocate into the nucleus andregulate gene expression by binding to Smad-specific binding elements inthe promoters of TGF-β-regulated genes. In humans, TGF-β overexpressionhas been detected in many cancer types and correlates with tumormetastasis, progression and prognosis. Many studies have indicated thatTGF-β can function as a tumor suppressor and promoter depending on thecontext. TGF-β acts as a tumor suppressor by inhibiting cellproliferation, while as a tumor promoter, TGF-β induces anepithelial-mesenchymal transition (EMT), cell motility and invasion.

EMT has been recognized as a key process for embryonic development andmetastasis. Cells undergoing EMT down-regulate the expression of theE-cadherin epithelial marker and increase the expression of N-cadherin,a mesenchymal marker. This process has been shown proceed through a setof transcription factors including the Snail and Slug zinc-fingerproteins, the Twist bHLH factor and the ZEB1 zinc-finger protein. TGF-βis a potent inducer of EMT, which was first recognized in culturednormal mammary epithelial cells. TGF-β can induce EMT by activatingSmad-dependent and Smad-independent pathways. Ectopic expression ofSmad2 or Smad3 with Smad4 enhances EMT, whereas ectopic expression ofdominant-negative Smad2, Smad3 or Smad4 blocks TGF-β-induced EMT.

TGF-β acts as a tumor suppressor in the early stages of cancerprogression, and it becomes a tumor promoter in later stages. TGF-β1,TGF-β2 and TGF-β3 overexpression has been reported in human ovariantumors. Ovarian cancer is thought to arise from normal ovarian surfaceepithelium (OSE). TGF-β has been shown to inhibit human OSEproliferation and induce apoptosis, which may prevent theover-proliferation of cells during a normal ovulatory cycle. In thelater stages of ovarian cancer, TGF-β enhances tumor cell proliferationand promotes metastasis by inducing an EMT.

It has recently been recognized that high-grade serous ovarian carcinoma(HGC) and low-grade serous ovarian carcinoma (LGC) are fundamentallydifferent types of tumors that develop from distinct molecular pathways.Compared with HGC, LGC accounts for a small proportion (9%) of allserous ovarian carcinomas. Invasive LGC is developed from non-invasiveborderline serous ovarian tumors (SBOT). In ovarian cancer,TGF-β-induced EMT is believed to play an important role in theregulation of cell invasion and metastasis. It has been shown that TGF-βand TβRII are expressed in primary human borderline ovarian tumors.Recent studies demonstrate that E-cadherin down-regulation induces SBOTcell invasion, suggesting that EMT is involved in the progression fromnon-invasive SBOT to invasive LGC and that TGF-β induces SBOT invasionby activating EMT (Cheng J-C, (2012) TGF-Beta Induces Serous BorderlineOvarian Tumor Cell Invasion by Activating EMT but Triggers Apoptosis inLow-Grade Serous Ovarian Carcinoma Cells. PLoS ONE 7(8): e42436.doi:10.1371).

PEO1 is an adherent cell line derived from a malignant effusion from theperitoneal ascites of a patient with a poorly differentiated serousadenocarcinoma. Cisplatin-sensitive ovarian cancer cell line PEO1 and-resistant PEO4 were established from the same patient before treatmentand after developing resistance to platinum-based chemotherapy. PEO1 andPEO4 express mRNA for IGF-I and mRNA for the IGF type I, IGF type II andinsulin receptors; the presence of type I IGF receptors was confirmed byimmuno-cytochemistry. IGF-I and insulin markedly stimulated theproliferation of PEO1 and PEO4. Both expressed mRNA for TGF beta 1 and 3(Bartlett et al. Brit J. Cancer. 1992 May; 65(5):655-60). The TGF betareceptor pathway is also altered in these cells.

5.5.8 Anti-Angiogenic Effects of Grp94 Inhibitors

Recently, Finotti et al demonstrated that Grp94 promotes the angiogenictransformation of Human Umbilical Vein Endothelial Cells (HUVECs) by acytokine-like mechanism, and that this effect is more pronounced whenGrp94 is in complexes with human IgG (Tramentozzi et al., 2008;Tramentozzi et al., 2009). A similar, strong angiogenic-transformingproperty has been observed with complexes of IgG with Grp94 purifiedfrom the plasma of type 1 diabetic subjects, a finding that indirectlyproved the capacity of these complexes to promote and sustain in vivothe inflammatory reactions predicting the development of stable vascularalterations. As discussed below, we show that the proliferative andangiogenic-transforming capacity of Grp94 was affected by the Grp94inhibition.

Both native Grp94 and the IgG-containing fraction purified from plasmaof diabetic subjects, referred further as peak 2, were tested incultures of HUVECs in both absence and presence of the Grp94 inhibitorPU-H54. As shown in FIG. 18b , morphological changes observed with Grp94and peak 2 resembled those typically characterizing the differentiationof endothelial cells into capillary-like structures, in which longcytoplasmic protrusions of enlarged cells connect with each other toborder the cavity of new tubes, interspersed with clusters of smallercells. PU-H54 especially at the highest concentration of 10 μM was ableto change the angiogenic-like transformation of HUVECs induced by Grp94and by peak 2. Overall, the morphologic changes observed in the presenceof PU-H54 show that PU-H54 at its IC₅₀ displays an anti-angiogeniceffect on HUVECs while it does not affect substantially the cellproliferation (see FIG. 18a ).

5.5.9 Inflammatory Diseases

Toll-like receptors (TLRs) play an important role in inflammatoryresponses. Grp94 chaperones multiple TLRs, and is required for thefunction of these receptors. TLR9 detects an un-methylated DNA and isknown to play a role in systemic lupus erythematosus or rheumatoidarthritis. A recent study demonstrated a role for Grp94 in TLR9stability and conformation. Based on experiments described below, wehave found that the Grp94 inhibitors of the disclosure are capable ofmodulating inflammatory responses through the inhibition of the Grp94chaperoning of Toll-like receptors (TLRs), particularly TLR9. Inparticular embodiments, the Grp94 inhibitors of the disclosure can beused in the treatment of inflammatory diseases such as lupuserythematosus and rheumatoid arthritis.

In order to evaluate the effect of Grp94 inhibitors on TLR9 response tostimulus, we treated cells with selective Grp94 inhibitors PU-WS13 (FIG.19a ) and PU-H54 (FIG. 19b ). The TLR9 ligand, CpG DNA, induced TNF-αproduction from mouse macrophages (RAW 264.7). Treatment with PU-WS13(FIG. 19A) and PU-H54 (FIG. 19B) inhibited this response in aconcentration dependent manner. Treatment with vehicle alone did notinhibit signaling (not shown). Hence, these studies suggest selectiveGrp94 inhibitors improve inflammatory symptoms in diseases in which TLR9plays a role.

5.6 Fluorescence Polarization Assays

The disclosure provides a versatile experimental fluorescencepolarization (FP) assay that can test rapidly and accurately the bindingaffinity of all major Hsp90 paralogs and has a testing range that spanslow nanomolar to millimolar binding affinities.

5.6.1 Development of FP Probes

Most assays published to date and used to assess binding of smallmolecules to the four Hsp90 paralogs, such as intrinsic tryptophanfluorescence, affinity-resin competitive binding and isothermaltitration calorimetry, are laborious and costly as they make use ofsignificant amounts of proteins. For the cytosolic Hsp90, the FP assayhas become one of the most extensively used to identify and test Hsp90inhibitors. There are numerous reasons why FP is an ideal method formeasuring protein-ligand interactions and why it has become a favoritetool for Hsp90. First, it is a quick, homogeneous, i.e. there is nonecessity for separation of free and bound ligand, high reproducibilityand facility for automation assay. By simply mixing a protein with afluorescently labeled ligand, FP is able to measure real-timeprotein-ligand interactions in solution where binding of thefluorescently labeled ligand, also referred to as an FP probe, to aprotein results in increased polarization values and is directlyproportional to the fraction of bound ligand. Its theory, firstdescribed in 1926 by Perrin, is based on the observation thatfluorescent molecules in solution, excited with plane-polarized light,will emit light back into a fixed plane (i.e. the light remainspolarized) if the molecules remain still during the excitation of thefluorophore. Molecules, however, rotate and tumble and the planes intowhich light is emitted differ from the plane used for initialexcitation. Nonetheless, upon binding of the small probe to a protein(i.e. a large, slowly rotating molecule) motility is reduced leading tohigher FP. Unlabeled ligands that bind to the protein will compete withthe probe, leading to lower FP. FP therefore provides a direct readoutof the extent of probe binding to a protein. Second, FP is also wellsuited for Hsp90 because it is an assay that requires no engineering ofthe protein. As reported, Hsp90 is a highly flexible molecular chaperonewhose function is very sensitive to interference with its conformationalmodality, such as the attachment of labels may lead to, and thus, FP isbest suited for this class of proteins. Third, there are numerous Hsp90inhibitor chemotypes for which extensive chemistry has been developedand binding to Hsp90 revealed by crystallography, and therefore thechoice for the FP probe and knowledge over the site of its fluorescencelabeling is available. To date however, no FP assay that efficientlytests for affinity and selectivity of small molecules to all four Hsp90paralogs has been reported.

Several FP tracers for Hsp90 that bind to the NBD have been reported andinclude a number based on geldanamycin (GM), GM-BODIPY, GM-Cy3b, and twocarboxyfluorescein (FAM) probes based on the pyrazole scaffold(VER-00045864 and VER-00051001. More recently, an FP probe of aderivative of Sansalvamide A-amide has been reported, which, in contrastto other probes binds Hsp90 at the N-terminal/middle domains.

None of the aforementioned probes have been systematically assessed fortheir suitability as tracers in FP assays with each of the paralogs.While the GM and pyrazole probes have been extensively used to measurebinding to Hsp90π, Hsp90β as well as to total Hsp90 in a cancer celllysate, their use in measuring binding to Grp94 and Trap-1 is morelimited. In our own hands we have found GM-Cy3b to be inadequate as atracer for Trap-1. In order to get a suitable assay window, its userequires a considerable amount of protein and is therefore less suitablefor large structure-activity relationship studies.

We therefore proceeded here to design FP probes based on the Hsp90inhibitor PU-H71 labeled through different linkers with fluorescein(FITC). We hypothesized that due to its known binding mode andwell-established chemistry, useful FP probes amenable forparalog-selectivity testing may be created around this ligand. Compound40, a FITC derivative of PU-H71 with optimal properties for flowcytometry and fluorescence microscopy, was also included in our analysis(FIG. 20). See Taldone, T.; Gomes-DaGama, E. M.; Zong, H.; Sen, S.;Alpaugh, M. L.; Zatorska, D.; Alonso-Sabadell, R.; Guzman, M. L.;Chiosis, G. Synthesis of purine-scaffold fluorescent probes for heatshock protein 90 with use in flow cytometry and fluorescence microscopy.Bioorg. Med. Chem. Lett. 2011, 21, 5347-5352

The linker and its attachment mode, both important in the synthesis ofFP chemical probes because they can affect binding to the targetprotein, can be predicted for PU-H71 from the available structuralstudies. For the preparation of a suitable FP probe it is also importantthat the linker not be excessively long or flexible because of thepropeller effect. Depolarization due to flexibility in the attachment ofthe dye, referred to as the “propeller effect”, distorts therelationship between fluorescence polarization and molecular weight. Forthis reason, it is generally preferable to use dyes without longaliphatic linkers between the fluorophore and the reactive group in thepreparation of fluorescence polarization assay probes.

In order to determine a linker length optimal for binding to all Hsp90paralogs, we docked the linker-modified PU-H71 ligands into therespective paralogs of Hsp90, i.e. Hsp90a (PDB ID: 2FWZ_ENREF_32(Immormino, R. M.; Kang, Y.; Chiosis, G.; Gewirth, D. T. Structural andquantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90inhibitors. J. Med. Chem. 2006, 49, 4953-4960.)), Hsp90β (PDB ID: 3NMQ(Yun, T. J.; Harning, E. K.; Giza, K.; Rabah, D.; Li, P.; Arndt, J. W.;Luchetti, D.; Biamonte, M. A.; Shi, J.; Lundgren, K.; Manning, A.;Kehry, M. R. EC144, a Synthetic Inhibitor of Heat Shock Protein 90,Blocks Innate and Adaptive Immune Responses in Models of Inflammationand Autoimmunity. The Journal of Immunology 2011, 186, 563-575)), Grp94(PDB ID: 3O2F, 2EXL (Immormino, R. M.; Metzger, L. E.; Reardon, P. N.;Dollins, D. E.; Blagg, B. S.; Gewirth, D. T. Different poses for ligandand chaperone in inhibitor-bound Hsp90 and GRP94: implications forparalog-specific drug design. J. Mol. Biol. 2009, 388, 1033-1042)) andTrap-1 (Homology Model). FITC was covalently bonded to the N-9 positionof the purine-scaffold via at least a three carbon linker as this wouldorient the probe towards solvent without affecting binding to the targetprotein (FIG. 21a ). A shorter linker would lead to clashes between theprobe and a leucine residue positioned in all paralogs at the exit ofthe binding site (Leu107 in Hsp90a and Hsp90β, Leu163 in Grp94 andLeu172 in Trap-1) (FIG. 21b for Hsp90α).

To determine the optimal chain length for FP properties we synthesized anumber of probes with linkers ranging from 3 to 8 carbons (Scheme 20).These were prepared by a three-step sequence from 112, commencing withN9-alkylation with ω-bromophthalimides to yield 113a-113d (Scheme 20).Following unmasking of the amine with hydrazine and attachment of FITC,114a-114d were obtained after HPLC purification (Scheme 20).

5.6.2 Binding of FP Probes to Hsp90 Paralogs

The synthesized FITC-derivatives were first evaluated for theirpotential as FP tracers for binding to Hsp90 in a cancer cell homogenate(FIG. 22a ). The potential tracers were initially evaluated by titrationwith increasing amounts of lysate up to 50 μg of total protein (FIG. 22a). To be useful in FP, the binding affinity of the probe should be highand the binding range (i.e. assay window), defined as the mP value atsaturation minus the mP recorded for probe alone should be large. Asobserved in FIG. 22a , as the amount of lysate, and thus of Hsp90,increased so did the assay window. Good performance was observed for allprobes, with an excellent assay window of >100 mP for Compound 115a.Similar to other Hsp90 FP assay probes, when measured at 4° C. tomaintain proper folding of Hsp90, the binding assay between Compound115a and Hsp90 reached equilibrium by 8 h and remained stable for morethan 24 h (not shown). While Compound 115a, the analog with a 3-carbonlinker, was optimal, Compound 40, the N-isopropyl analog of Compound115a, and Compounds 115b and 115c, the 4- or 6-carbon linker compoundsrespectively, performed acceptably well (FIG. 22a ).

We next determined, in a standard saturation binding experiment thatmeasures ligand binding in the presence of varying concentration ofprotein, the ability of these ligands as probes for the Hsp90 paralogs(FIGS. 22b-c ). Taken as a whole, saturation binding experiments withCompound 115a showed it to be an excellent tracer for each Hsp90 paralogwith an assay window of >150 mP and an apparent K_(d)=1.4, 1.6, 6.6, and5.9 nM for Hsp90π, Hsp90β, Grp94 and Trap-1, respectively (FIG. 22b )and we proceed further here to use it as a probe in evaluating theparalog-selective binding of Hsp90 inhibitors and Hsp90 endogenousligands. Interestingly, Compound 40 showed a 1-log preference forHsp90π, Hsp90β and Trap-1 over Grp94 (apparent K_(d)=3.9, 2.8, 30.7, and5.8 nM for Hsp90π, Hsp90β, Grp94 and Trap-1, respectively) with a poorassay window, i.e. less than 100 mP, for Grp94 (FIG. 22c ).

5.6.3. Suitability of the FP Assay for Evaluating the Selectivity andAffinity of Small Molecules for the Hsp90 Paralogs

Having found a probe that binds effectively to all four Hsp90 paralogs,we next validated its ability to evaluate paralog affinity andselectivity for small molecule ligands. Specifically, we evaluated thebinding of ATP and ADP, the two endogenous Hsp90 paralog ligands, forwhich paralog binding affinity has been extensively explored by means ofintrinsic tryptophan fluorescence and isothermal titration calorimetry.We observe for these ligands relative affinities in line with what hasbeen reported previously for each paralog (Table 13). Specifically, theADP interaction with Hsp90 was reported to be much tighter than that ofATP (41 μM versus 840 μM), (McLaughlin, S. H.; Ventouras, L. A.;Lobbezoo, B.; Jackson, S. E. Independent ATPase activity of Hsp90subunits creates a flexible assembly platform. J. Mol. Biol. 2004, 344,813-826) which is very much in line with our findings (Table 13). ADPwas reported to be a slightly weaker binder of Hsp90a than of Hsp90 (51μM versus 34 μM), (Richter, K.; Soroka, J.; Skalniak, L.; Leskovar, A.;Hessling, M.; Reinstein, J.; Buchner, J. Conserved conformationalchanges in the ATPase cycle of human hsp90. J. Biol. Chem. 2008, 283,17757-17765), which is what we find (59 μM versus 42 μM, Table 13).Additionally, as previously reported, we show Grp94 and Trap-1 to showlittle discrimination between both nucleotides. Grp94 binds bothnucleotides relatively well, with binding affinities reported rangingfrom 2.3 to 3.4 μM to 5 μM (Frey, S.; Leskovar, A.; Reinstein, J.;Buchner, J. The ATPase cycle of the endoplasmic chaperone Grp94. J.Biol. Chem. 2007, 282, 35612-35620), which compares well with 3.2 μM and11.4 μM we record for ATP and ADP, respectively (Table 13). As in ourstudy, ATP was found to be a slightly tighter binder of Grp94 than ADP.Trap-1, which most closely resembles the bacterial Hsp90, HtpG, isreported to bind ATP with approximately 10-fold greater affinity thandoes Hsp90.

We next used the newly developed FP assay to evaluate the paralogaffinity and selectivity of Hsp90 NBD inhibitors encompassing a varietyof chemical classes (FIG. 23). All but GM have been or still are inclinical evaluation for cancers. The results for each paralog of Hsp90are summarized in Table 13 and show that all inhibitors effectivelycompete with Compound 115a, demonstrating specificity of binding for theprobe. The low nanomolar binding affinities for Hsp90α/β that wemeasured for these inhibitors correlate well with their biologicalactivity determined in several cancer cells.

TABLE 13 Hsp90 paralog affinity determined for Hsp90 inhibitors inclinical development using 43a as a FP probe. The paralog bindingaffinity of Hsp90-regulatory nucleotides is also presented. Hsp90αHsp90β Grp94 Trap-1 IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM) GM 28 22 10661 17-AAG 46 45 31 1,496 BIIB021 19 17 124 90 PU-H71 43 42 30 205CUDC-305 33 38 190 1,586 SNX-2112 29 25 578 726 NVP-AUY922 20 16 12 38STA-9090 5 5 10 51 ADP 59,308 42,159 11,447 55,594 ATP 861,330 893,6773,241 31,303

Interestingly, while it is believed that the clinical Hsp90 inhibitorsbind equally well to all paralogs, we determined a spectrum of paralogbinding preferences (Table 13). Of note, all these inhibitors boundapproximately equally well and with low nanomolar affinity to thecytosolic Hsp90s, as indicated previously by the extensive interactionsthey form with the pocket.

In contrast, we found a significant difference among the several agentswith regards to their affinity for Grp94 and Trap-1. Most striking wasan almost 2-log loss of affinity for Trap-1 recorded for 17-AAG andCUDC-305/Debio092 (Hsp90 vs Trap-1: 46 nM vs 1.5 μM for 17-AAG, 35 nM vs1.5 M for CUDC-305/Debio092). Lower binding efficacy for Trap-1 was alsoseen for the other agents, with a decrease ranging from 25-fold for GMand SNX-2112, 10-fold for STA-9090 to 5-fold for BIIB021 and PU-H71 and2-fold for NVP-AUY922. The affinity of these agents for Grp94, whilecomparable to Hsp90 for most agents, was substantially lower for a fewinhibitors. Specifically, an approximately 10-fold loss of affinity wasnoted for BIIB021, CUDC-305 and SNX-2112 (Hsp90 vs Grp94: 19 nM vs 124nM, 33 nM vs 190 nM and 29 nM vs 578 nM, respectively).

TABLE 14 Solubility of WS-13 salts in water. Type of Salt SolubilityAppearance pH Hydrochloride Mono 6.25 mg/mL Clear solution 5.0 Di 12.5mg/mL Clear solution 3.5 Mesylate Mono 25.0 mg/mL Clear solution 6.5 Di50.0 mg/mL Clear solution 3.0 Lactbionate Mono 6.25 mg/mL Clear solution6.5 Di 25.0 mg/mL Clear solution 4.5 Succinate Mono <5.0 mg/mL InsolubleDi  5.0 mg/mL Hazy solution Citrate Mono <5.0 mg/mL Insoluble Di  9.4mg/mL Hazy solution Mesylate salt of WS-13 had higher solubility inwater.

TABLE 15 Solubility of WS-13 salts in Phosphate Buffer Saline (PBS, pH =7.4). Type of Salt Solubility Appearance pH Hydrochloride Mono <5.0mg/mL Insoluble Di <5.0 mg/mL Insoluble Mesylate Mono   5.0 mg/mL Clearsolution 6.6 Di 12.5 mg/mL Clear solution 3.5 Lactbionate Mono <5.0mg/mL Hazy solution 3 Di   5.0 mg/mL Clear solution Succinate Mono <5.0mg/mL Insoluble Di <5.0 mg/mL Hazy solution Citrate Mono <5.0 mg/mLInsoluble Di <5.0 mg/mL Insoluble Oxalate Mono <5.0 mg/mL Insoluble Di  9.4 mg/mL Hazy solution Tosylate Mono <5.0 mg/mL Slight Hazy solutionDi   9.4 mg/mL Slight Hazy solution Phosphate Mono <5.0 mg/mL InsolubleDi <5.0 mg/mL Insoluble Tartarate Mono <5.0 mg/mL Insoluble Di <5.0mg/mL Hazy solution Maleate Mono <5.0 mg/mL Insoluble Di <5.0 mg/mLInsoluble Acetate Mono <5.0 mg/mL Insoluble Di <5.0 mg/mL InsolubleTrifluoroacetate Mono <5.0 mg/mL Insoluble Di <5.0 mg/mL InsolubleMesylate salt of WS-13 had higher solubility in PBS.

EXAMPLES

6.1 Materials and Methods

Biochemical and Cellular Assays.

Expression and phosphorylation of proteins was analyzed byimmunoblotting. Chemical precipitation and immunoprecipitation assayswere performed to determine the interaction between the Hsp90 paralogsand proteins. Analysis of cell cycle and of cell surface expression ofGrp94 was carried out by flow cytometry.

X-Ray Structure Determination.

Complexes were formed by adding a 2-3 fold molar excess of PU-H54 to aconcentrated Hsp90 αN or Grp94N solution prior to crystallization. TheHsp90 and Grp94 complex structures were determined by X-ray diffractionto a resolution of 1.5 Å and 2.0 Å, respectively, and were solved bymolecular replacement.

Molecular Modeling.

All computations were carried out on a HP workstation xw8200 with theUbuntu 8.10 operating system. The protein structure was prepared usingthe protein preparation wizard in the Schrodinger software graphicaluser interface Maestro (version 8.5). Protein sequences and crystalstructures were downloaded from the NCBI (www.ncbi.nlm.nih.gov) and theRCSB (www.rcsb.org) database, respectively. The Trap-1 homology modelwas constructed in Prime (version 2.0) and the crude homology model wasfurther refined by minimization using Macromodel (version 9.6). SiteMap(version 2.2) analysis was performed on the protein structures, asindicated. All docking studies were performed with Glide (version 5.0).

Statistical Analysis.

The results were analyzed by unpaired 2-tailed t-tests in Prism5(GraphPad). Data are presented as the mean±SD or SEM of duplicates ortriplicates. Error bars represent the mean SD or SEM. When a singlepanel is presented it is representative of two or three individualexperiments.

Reagents.

Recombinant Hsp90a (ADI-SPP-776), Hsp90β (ADI-SPP-777) and Trap-1(ADI-SPP-848) were purchased from Enzo Life Sciences. Grp94 wasgenerated as previously reported (Dollins, D. E., Immormino, R. M. &Gewirth, D. T. Structure of unliganded GRP94, the endoplasmic reticulumHsp90. Basis for nucleotide-induced conformational change. J. Biol.Chem. 280, 30438-47 (2005); Dollins, D. E., Warren, J. J., Immormino, R.M. & Gewirth, D. T. Structures of GRP94-nucleotide complexes revealmechanistic differences between the hsp90 chaperones. Mol. Cell 28,41-56 (2007)). The synthesis and characterization of the purine-scaffoldcompounds and chemical tools was reported elsewhere (Llauger, L. et al.Evaluation of 8-arylsulfanyl, 8-arylsulfoxyl, and 8-arylsulfonyl adeninederivatives as inhibitors of the heat shock protein 90. J. Med. Chem.48, 2892-905 (2005); He, H. et al. Identification of potent watersoluble purine-scaffold inhibitors of the heat shock protein 90. J. Med.Chem. 49, 381-90 (2006); Moulick, K. et al. Affinity-based proteomicsreveal cancer-specific networks coordinated by Hsp90. Nat. Chem. Biol.7, 818-26 (2011). Geldanamycin was purchased from Sigma-Aldrich andlapatinib from Selleck Chemicals. The synthesized compounds were fullycharacterized and structures confirmed by direct comparison to previousreports and determined to have a purity of >98%.

Cell Lines.

The HER2 overexpressing breast cancer cells SKBr3, BT474, MDA-MB-361,MDA-MB-453 and AU565, as well as the low HER2 breast cancer cells MCF7,BT20 and MDA-MB-231, were obtained from the American Type CultureCollection (ATCC). Cells were cultured routinely in McCoy's 5A (10% FBS,SKBr3), DME/F12 (10% FBS, BT474 and MDA-MB-231), RPMI (10% FBS, AU565),MEM (10% FBS, MCF7 and BT20) and L-15 (20% FBS, MDA-MB-361 andMDA-MB-453) supplemented with 1% Glutamax and 1% penicillin andstreptomycin (Pen/Strep). C2C12 and HEK293 cells were purchased fromATCC and cultured in DMEM in the presence of 10% FBS and 1%penicillin/streptomycin. Gastric carcinoma cell lines OE19, NCI-N87 andMKN74 were grown in DME media (MKN74), or RPMI media supplemented with10% FBS and 1% penicillin/streptomycin. Ovarian cancer cell lines PEO-1,PEO-4, OV-1847, OVCAR4 and A2780, Ewing's sarcoma cell lines TC71 andA673 were generous gift from Dr. Malcolm A. S. Moore lab. All cell lineswere grown in M-5 media supplemented with 10% FBS, 1% GlutaMax (Gibco,cat #35050-061) and 1% penicillin/streptomycin. Pancreatic cancer celllines PANC-1, CAPAN2 and CFPAC were purchased from ATCC and grown in DME(PANC-1), McCoy's 5a Medium Modified (CAPAN-2) and IMDM mediasupplemented with 10% FBS and 1% penicillin/streptomycin. Breast cancercell lines HCC 1806, MDA-MB-231 and MDA-MB-468 were purchased from ATCCand grown in RPMI (HCC1806) and DME (MDA-MB-231 and MDA-MB-468) mediasupplemented with 10% FBS and 1% penicillin/streptomycin. When cultured,cells in L-15 medium were kept in a humidified atmosphere without CO2 at37° C. and all other cell lines were incubated in the humidified cellincubators with CO2 at 37° C.

Crystallization of Grp94 and hHsp90 PU-H54 Complexes.

Recombinant canine Grp94N A41 and human Hsp90aN were purified asdescribed previously. Prior to crystallization, protein-inhibitorcomplexes were formed by the addition of a two-fold molar excess ofPU-H54 to Grp94 or a three-fold molar excess of PU-H54 to human Hsp90 at30 mg/ml in 10 mM Tris, pH 7.6, 100 mM NaCl, and 1 mM DTT. Grp94crystals were grown by hanging-drop vapor diffusion at 18° C. by mixinga 1:1 ratio of protein to reservoir solution containing 14-17%isopropanol, 300-375 mM MgCl2, 0.1-1.0% glycerol, and 100 mM Hepes, pH7.4. Grp94 crystals were cryo-protected by rapid passage through asolution containing 30% glycerol, 5% isopropanol, and 100 mM Hepes, pH7.4 before flash freezing in liquid nitrogen. Hsp90 crystals were grownby hanging-drop vapor diffusion at 4° C. by mixing a 1:1 ratio ofprotein to reservoir solution containing (11-15% PEG 2K MME, 200 mMMgCl2, and 100 mM sodium cacodylate, pH 6.5). Hsp90 crystals werecryo-protected by sequentially passing through reservoir solutionrapidly followed by a cryoprotectant solution containing 35% PEG 2K MME,200 mM MgCl2, and 100 mM sodium cacodylate, pH 6.5 before flash freezingin liquid nitrogen.

Data Collection, Structure Determination and Refinement.

X-ray diffraction data for the Grp94NA41+PU-H54 and human Hsp90N+PU-H54co-crystals were collected on a MAR-325 CCD detector at SSRL beamline11-1 using an X-ray wavelength of 0.979 Å. Data were indexed and scaledusing HKL2000. Initial phases for the co-crystals were obtained bymolecular replacement using Phaser software in the CCP4 software suite.The search model for Grp94NA41 was the core region (residues 100-166 and200-337) of Grp94NA41+ATP (PDB ID 1TC0), and the search model for hHsp90was Hsp90+PU-H71 (PDB ID 2FWZ). Initial molecular replacement modelswere manually rebuilt in Coot and refined using Refmac 5.5 in CCP4.Ligand topology files for PU-H54 were generated using the Dundee PRODRGserver. For the Grp94NA41+PU-H54 structure, density modification wascarried out using DM software in CCP4 and TLS parameters generated usingTLSMD were applied in the final stage of refinement. Final modelscontained no Ramachandran outliers, and 95.1 and 97.6% of the residuesfell in Ramachandran favored regions for the Grp94NA41+PU-H54 andHsp90N+PU-H54 structures, respectively.

Sequence Alignment.

Sequences were aligned and shown as Percentage Identity view using theprogram of T-Coffee Multiple Sequence Alignment in Jalview 2.7(http://www.tcoffee.org/Projects/tcoffee/).

Homology Model for Trap-1.

The protein structures of the Hsp90a NTD (PDB ID: 2FWZ), Grp94 NTD (PDBID: 302F) and the amino acid sequence of Trap-1 protein (Accessionnumber: Q12931) were used for model building. To create the model, theprotein sequence of Trap-1 protein (Accession number: Q12931) wasentered as an input sequence in Prime's Structure Preparation wizard.The homologous protein Hsp90a (PDB ID: 2FWZ) with 31% identities, 47%positives, 20% gaps and Grp94 (PDB ID: 302F) with 28% identities, 45%positives and 28% gaps were imported. The NTD Trap-1 sequence and thetemplates were aligned and then edited using parameters as implementedin Prime. In the “Build structure” option of Prime, amino acids 179-196(Grp94) were selected from PDP ID 302F whereas the remaining amino acidsfrom Hsp90a (PDB ID: 2FWZ). The structure was then built using selectedsequence alignment of the template(s), taking solvent, ligand, forcefield, and other contributions into account via a series of algorithmsimplemented in Prime. Structural discontinuities were optimized byinserting template gaps for more than twenty residues. All loops wererefined with the default parameter settings of Prime. The obtainedhomology model of Trap-1 was further refined using the proteinpreparation wizard available in Maestro (version 8.5). Partial atomiccharges were assigned according to the OLPS-AA force field. To obtain amore reliable 3D structure of Trap-1, the homology model was furthersubjected to a series of energy minimization steps that consisted of5,000 iterations of steepest descent (SD) and conjugate gradient (CG),until the root mean-square deviation (rmsd) was lower than 0.001 kcalmol⁻¹ Å⁻¹.

Ligand Preparation.

All the compounds were constructed using the fragment dictionary ofMaestro (version 8.5). The geometry of compounds was optimized using theMacromodel program (version 9.6) and the OLPS-AA force field⁷. Resultingligands were further prepared using Ligprep (version 2.2) utilityprovided by Schrödinger LLC., New York.

Docking.

The x-ray crystal structure of Hsp90a NTD in complex with PU-H71 (PDBID: 2FWZ), Hsp90β NTD in complex with EC44 (PDB ID: 3NMQ), Grp94 NTD incomplex with PU-H54 (PDB ID: 3O2F), ADP (PDB ID: 1TC6) and unliganded(PDB ID: 1YT0) and Trap-1 homology model were first aligned using theprotein structure alignment tool, then were optimized for subsequentgrid generation and docking using the default parameters in ProteinPreparation Wizard provided by Schrödinger LLC. Grids were then preparedusing the Receptor Grid Generation tool in Glide (version 5.0)(Friesner, R. A. et al. Glide: a new approach for rapid, accuratedocking and scoring. 1. Method and assessment of docking accuracy. J.Med. Chem. 47, 1739-49 (2004); Halgren, T. A. et al. Glide: a newapproach for rapid, accurate docking and scoring. 2. Enrichment factorsin database screening. J Med Chem 47, 1750-9 (2004); Friesner, R. A. etal. Extra precision glide: Docking and scoring incorporating a model ofhydrophobic enclosure for protein-ligand complexes. J. Med. Chem 49,6177-6196 (2006)) Next, the extra precision (XP) Glide docking methodwas used to dock compounds flexibly into the ATP binding site of theHsp90 paralogs. Upon completion of each docking calculation, at most 100poses per docking were run and at most 10 poses per ligand were allowedto be generated. Top-scored docking poses (orientation plusconformation) based on the Glide scoring (GScore) function wereanalyzed. To validate docking parameters and experimental set-up,endogenous ligands (PU-H71, PDB ID: 2FWZ; EC44, PDB ID: 3NMQ; PU-H54,PDB ID: 302F; ADP, PDB ID: 1TC6) were removed from the binding site andre-docked. Very good agreement was found between inhibitor pose asobtained from docking analyses and as captured in the crystal structure(RMSD of 0.7 Å; PDB ID: 2FWZ, 0.9 Å; PDB ID: 3NMQ, 0.04 Å; PDB ID: 302Fand 1.2 Å; PDB ID: 1TC6) between the predicted conformation and theobserved x-ray crystallographic conformation, validating the dockingstrategy.

Binding Site Analysis:

SiteMap (Halgren, T. A. Identifying and Characterizing Binding Sites andAssessing Druggability. J. Chem. Information and Modeling 49, 377-389(2009); Halgren, T. New method for fast and accurate binding-siteidentification and analysis. Chemical Biology & Drug Design 69, 146-148(2007)) analysis was carried out on the x-ray crystal structures ofHsp90a (PDB ID: 2FWZ), Hsp90β (PDB ID: 3NMQ) and Grp94 (PDB ID: 302F)and the refined homology model of Trap-1 using “Evaluate a singlebinding site region” using default parameters implemented in SiteMap(version 2.2). Next, to investigate the ATP binding site, hydrophobicand hydrophilic contour maps were constructed using default parametersas implemented in the “Manage surfaces” function.

Hsp90 Saturation Binding Assay.

The Hsp90 FP saturation assays were performed on an Analyst GTinstrument (Molecular Devices, Sunnyvale, Calif.) and carried out inblack 96-well microplates (Corning #3650) in a total volume of 100 μL ineach well. A stock of 10 μM 40 or 115a-115d was prepared in DMSO anddiluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT,5 mM MgCl₂, 20 mM Na₂MoO₄, and 0.01% NP40 with 0.1 mg/mL BGG). Todetermine the equilibrium binding of 112 or 115a-115d, increasingamounts of Hsp90π, Hsp90β, Grp94 or Trap-1 (up to 250 nM), or SKBr3lysate (up to 50 μg of total protein) were incubated with 3 nM of 40 or115a-115d. The assay plate was incubated on a shaker at 4° C. for theindicated times and the FP values in mP were measured. The assay windowwas calculated as the difference between the FP value recorded for thebound fluorescent tracer and the FP value recorded for the freefluorescent tracer (defined as mP−mP_(f)).

Fluorescence Polarization (FP) Measurements on Grp94 Inhibitors of theDisclosure.

The Hsp90 FP competition assays on Grp94 inhibitors of the disclosurewere performed as described below.

Fluorescence Polarization (FP) Measurements Using New Probes.

The Hsp90 FP competition assays were performed on an Analyst GTinstrument (Molecular Devices, Sunnyvale, Calif.) and carried out inblack 96-well microplates (Corning #3650) in a total volume of 100 μL ineach well. A stock of 10 μM 112 or 115a-115d was prepared in DMSO anddiluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT,5 mM MgCl₂, 20 mM Na₂MoO₄, and 0.01% NP40 with 0.1 mg/mL BGG). To eachwell was added 3 nM fluorescent 40 or 115a-115d, protein (25 nM Hsp90π,25 nM Hsp90β, 25 nM Grp94, 30 nM Trap-1) or SKBr3 lysate (4.5 μg totalprotein), and tested inhibitor (initial stock in DMSO) in a final volumeof 100 μL HFB buffer. Compounds were added in triplicate wells. For eachassay, background wells (buffer only), probe controls (free, probe only)and bound probe controls (probe in the presence of protein or SKBr3lysate) were included on each assay plate. The assay plate was incubatedon a shaker at 4° C. for 24 h and the FP values in mP were measured. Thefraction of probe bound to Hsp90 was correlated to the mP value andplotted against values of competitor concentrations. The inhibitorconcentration at which 50% of bound probe was displaced was obtained byfitting the data. All experimental data were analyzed using SOFTmax Pro4.3.1 and plotted using Prism 4.0 (Graphpad Software Inc., San Diego,Calif.).

Cell Fractionation and Immunoblotting.

Cells were either treated with DMSO (vehicle) or indicated compounds for24 hr and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl,0.5% sodium deoxycholate and 0.5% NP40) supplemented with cocktailprotease inhibitors (Roche) to produce whole cell lysates. Lysates forcytosol and membrane fractions were harvested using ProteoExtractSubcellular Proteome Extraction Kit (Calbiochem) following themanufacturer's instructions. Protein concentrations were determinedusing BCA kit (Pierce) according to the manufacturer's instructions. Theprotein lysates (5-50 pg) were electrophoretically resolved by SDS-PAGE,transferred onto nitrocellulose membranes and probed with the indicatedprimary antibodies against: HER2 (Zymed, 28004), Hsp70 (Stressgen,SPA-810), Grp94 (Stressgen, SPA-850), Hsp90a (Abcam, Ab2928), Hsp90β(StressMarq, SMC-107B), Grp78 (Cell Signaling, 3183), Raf-1 (Santa Cruz,sc-133), phospho-Raf-1 (Cell Signaling, 9421), MEK1/2 (Cell Signaling,8727), phospho-MEK1/2 (Cell Signaling, 9154), ERK1/2 (Cell Signaling,4695), phospho-ERK1/2 (Cell Signaling, 4370), AKT (Cell Signaling,9272), GM130 (Cell Signaling, 2296), Flotillin 2 (Cell Signaling, 3436),Histone H4 (Cell Signaling, 2592), Histone H1 (Santa Cruz, sc-8030),Caspase 3 (Cell Signaling, 9665), cleaved PARP (Promega, G7341),a-tubulin (Sigma, T5168) and 3-actin (Sigma, A1978). After washing offthe excess antibodies, the membranes were incubated with thecorresponding horseradish peroxidase (HRP) conjugated secondaryantibody. Blots were visualized by autoradiography using the EnhancedChemiluminescence Detection System (GE Healthcare) according tomanufacturer's instructions. For all gels 3-actin was used as a proteinloading control.

Densitometry Analysis.

Films were scanned in Adobe Photoshop CS5 and quantitative densitometricanalysis was performed using ImageJ (NIH).

Protein Level Quantification.

In all instances when protein quantification was performed, proteinlevels were first normalized to 3-actin then to the levels of thevehicle only treated experimental conditions. All quantified proteinlevels are reported as a fraction of control (i.e. the value obtained inthe experimental condition was divided by the value obtained in thevehicle treated cells).

Chemical Precipitation (CP).

Agarose beads conjugated with Hsp90 inhibitors were washed three timeswith and finally suspended in Felts buffer (20 mM HEPES, 50 mM KCl, 5 mMMgCl2, 0.01% NP40, 20 mM Na2MoO4, pH 7.2˜7.3) (Moulick, K. et al.Affinity-based proteomics reveal cancer-specific networks coordinated byHsp90. Nat. Chem. Biol. 7, 818-26 (2011)). The bead conjugates (50 μL)were then incubated for 4 hrs at 4° C. with the indicated amounts ofcell lysate, and the volume was adjusted to 500 μl with Felts buffer.The complexes were then washed three times with Felts buffer andproteins in the pull-down were analyzed by immunoblotting. ForPU-WS13-biotin pull-down assays, the cell lysate was first incubatedovernight at 4° C. with biotinylated PU-WS13, then for 2 hrs with 50 μLHigh Capacity Streptavidin Beads (Thermosci). The beads were washedthree times with Felts buffer and the proteins in the pull-downsidentified by immunoblotting. Control beads containing2-methoxyethylamine, an Hsp90-inert molecule, or D-biotin were used tocontrol for non-specific binding.

Immunoprecipitation (IP).

The HER2 antibody (Cell Signaling, 2165), the Grp94 antibody (Abcam,Ab13509) or a normal rabbit IgG (Santa Cruz Biotechnology) wereincubated with the indicated amount of cell lysate and with 40 pLprotein A agarose beads (Roche). The mixture was incubated overnight ona rotator at 4° C. The beads were washed three times with RIPA bufferand separated by SDS-PAGE, followed by a standard immunoblottingprocedure.

Grp94 Depletion Assay.

The Grp94 antibodies (Abcam, Ab13509; Bioss, bs-0194R) or a normalrabbit IgG (Santa Cruz Biotechnology) were incubated with the indicatedamount of cell lysate and with 40 pL protein A agarose beads (Roche).The mixture was incubated for 4 hours on a rotator at 4° C. Thesupernatants were collected after centrifugation, then incubated withthe Grp94 antibody or a normal rabbit IgG and then with 40 μL protein Aagarose beads to further deplete Grp94 in the cell lysate. After threerounds of antibody depletions, the supernatants were collected andincubated overnight with the PU-WS13-biotin beads at 4° C. The beadswere washed three times with Felts buffer and separated by SDS-PAGE,followed by a standard immunoblotting procedure.

siRNA Knock-Down of Hsp90π, Hsp90β and Grp94.

Transient transfections were carried out by using Lipofectamine RNAiMaxreagent (Invitrogen, for SKBr3 cells) or electroporation with Neontransfection system (Life Technologies, for MCF7 cells.) according tomanufacturer's instructions. For each target, four different siRNAs werepurchased from Qiagen and designed against the open reading frame ofHsp90a (Gene Hsp90AA 1), Hsp90β (Gene Hsp90AB1) or Grp94 (Gene Hsp90B1).Control cells were transfected with scramble siRNA. Cells weretransfected with 20 nM siRNA and knock-down efficiency was evaluated atthe indicated time by immunoblotting. Electroporation in MCF7 wasoptimized and the experiments were performed using two 1230v 20 mspulses on Neon transfection system (Life Technologies). SKBr3 cells weretransfected with 20 nM siRNAs for 72 hrs, then re-transfected with 20 nMsiRNAs for another 48 hrs before WB analysis.

Kinase Screen.

For most assays, kinase-tagged T7 phage strains were grown in parallelin 24-well blocks in an E. coli host derived from the BL21 strain. E.coli were grown to log-phase and infected with T7 phage from a frozenstock (multiplicity of infection=0.4) and incubated with shaking at 32°C. until lysis (90-150 min). The lysates were centrifuged (6,000×g) andfiltered (0.2 μm) to remove cell debris. The remaining kinases wereproduced in HEK-293 cells and subsequently tagged with DNA for qPCRdetection. Streptavidin-coated magnetic beads were treated withbiotinylated small molecule ligands for 30 minutes at room temperatureto generate affinity resins for kinase assays. The liganded beads wereblocked with excess biotin and washed with blocking buffer (SeaBlock(Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand andto reduce non-specific phage binding. Binding reactions were assembledby combining kinases, liganded affinity beads, and test compounds in1×binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT).Test compounds were prepared as 40×stocks in 100% DMSO and directlydiluted into the assay. All reactions were performed in polypropylene384-well plates in a final volume of 0.04 ml. The assay plates wereincubated at room temperature with shaking for 1 hour and the affinitybeads were washed with wash buffer (lx PBS, 0.05% Tween 20). The beadswere then re-suspended in elution buffer (lx PBS, 0.05% Tween 20, 0.5 μmnon-biotinylated affinity ligand) and incubated at room temperature withshaking for 30 minutes. The kinase concentration in the eluates wasmeasured by qPCR. KINOMEscan's selectivity score (S) is a quantitativemeasure of compound selectivity. It is calculated by dividing the numberof kinases that bind to the compound by the total number of distinctkinases tested, excluding mutant variants. TREEspot™ is a proprietarydata visualization software tool developed by KINOMEscan. Kinases foundto bind are marked with red circles, where larger circles indicatehigher-affinity binding. The kinase dendrogram was adapted and isreproduced with permission from Science and Cell Signaling Technology,Inc.

Cell Viability Assessment.

Cells were treated for 72 h with the indicated inhibitors or transfectedfor 72 h with Grp94 siRNA or control siRNA and their viability wasassessed using CellTiter-Glo luminescent Cell Viability Assay (Promega)as previously described (Rodina, A. et al. Selective compounds defineHsp90 as a major inhibitor of apoptosis in small-cell lung cancer. Nat.Chem. Biol. 3, 498-507 (2007); Caldas-Lopes, E. et al. Hsp90 inhibitorPU-H71, a multimodal inhibitor of malignancy, induces complete responsesin triple-negative breast cancer models. Proc. Natl. Acad. Sci. USA 106,8368-73 (2009)). The method determines the number of viable cells inculture based on quantitation of the ATP present, which signals thepresence of metabolically active cells.

Immunofluorescence.

Cells were seeded and grown onto culture slides (BD Falcon) for 24 hrs.After washing with cold PBS, cells were fixed by treating at 4° C. for20 min with 4% paraformaldehyde in PBS, permeabilized with 0.1% TritonX-100 in PBS containing 10% FBS for 10 min, and blocked with 2% BSA for1 hr. After washing for four times with PBS, primary antibodies wereadded onto the chambers and cell were incubated overnight at 4° C.,washed again with PBS followed by incubation with the secondary antibodyfor 1 hr at room temperature. Cell were washed and then mounted andobserved under microscope (Leica Upright Confocal SP5). The primaryantibodies used in the assay are against: HER2 (Zymed, 28004), Grp94(Stressgen, SPA-850), Hsp70 (Stressgen, SPA-810), LAMP1-FITC (Abcam,ab25406), EEA1 (Abcam, ab70521), 58K Golgi-FITC (Abcam, ab27043) andCalnexin (BD, 610523).

Flow Cytometry.

Flow cytometry analysis was performed using MACSQuant analyzer (MiltenyiBiotec). 5×10⁴ to 5×10⁵ cells were seeded in 35 mm dishes andcentrifuged at 500 g for 5 mins. Excess medium was removed and the cellpellet was resuspended in cold medium containing human AB serum forblocking. Then the primary antibody Grp94-PE (Enzo, SPA-850PE) or anisotype control was added to each tube. Cells were incubated on ice for60 mins then washed with cold PBS. Cells were then stained on ice with7-AAD for 15 minutes and washed once with cold PBS. Cells were finallyresuspended in 1% paraformaldehyde and subjected to flow cytometryanalysis. Data were further analyzed by FlowJo (Ashland). Dead cellswith positive 7-AAD staining were excluded from the analysis. For theBrefeldin A trafficking assay, cells were treated with GolgiPlug (BDbiosciences, 555029) for 4 h according to the manufacturer'sinstructions. Cells were then either processed for live cell staining orfirst permeabilized with 0.1% Triton-X100 before flow analysis using theMACSQuant analyzer.

Assessment of Cell Surface Proteins.

Cell surface protein isolation kit (Pierce) was used to biotinylateproteins on the cell surface according to the manufacturer'sinstructions. Briefly, four 75 cm (Dollins, D. E., Warren, J. J.,Immormino, R. M. & Gewirth, D. T. Structures of GRP94-nucleotidecomplexes reveal mechanistic differences between the hsp90 chaperones.Mol. Cell 28, 41-56 (2007)) flasks of cells were incubated withSulfo-NHS-SS-biotin for 30 min at 4° C., the reaction was then quenchedand cells were lyzed. The biotinylated proteins were isolated usingNeutrAvidin Agarose beads, then eluted with Laemmli buffer and subjectedto SDS-PAGE analysis and immunoblotting. Alternatively, after biotinlabeling of the cell surface proteins, the biotinylated proteins werepurified by using monomeric Avidin beads, followed by elution ofproteins from the beads by incubation with 2 mM biotin for 6 hours at 4°C.

Cell Cycle and Apoptosis Assessment.

Cell cycle and apoptosis were assessed by flow cytometry after singlestaining with propidium iodide (PI, BD Pharmingen) or double stainingwith AnnexinV-FITC (BD Pharmingen) and 7AAD (BD Pharmingen),respectively. Specifically, for cell cycle analysis, cells were washedtwice with cold PBS and fixed in 70% ethanol overnight at 4° C. Fixedcells were collected at 1800 rpm for 10 min and stained with PBScontaining PI and DNase-free RNase A (Sigma-Aldrich) for 1 h at roomtemperature in the dark. DNA content was measured by BD LSRII flowcytometer and cell further analyzed using program of cell cycle analysisin FloJo (Ashland, Oreg.). The chicken erythrocyte nuclei singlets (CEN,Biosure) were used as the reference. For apoptosis assessment, livecells were collected, washed twice with cold PBS, resuspended in BindingBuffer and stained with AnnexinV-FITC and 7AAD for 15 min at roomtemperature in the dark. Signals from FL1 and FL3 channels werecollected by MACSQuant analyzer and further analyzed using FloJo. Earlyapoptosis was defined as AnnexinV+/7AAD-, and late apoptosis wasobserved as AnnexinV+/7AAD+.

C2C12 Differentiation and IGF-II Secretion Assay

(Wanderling, S. et al. GRP94 is essential for mesoderm induction andmuscle development because it regulates insulin-like growth factorsecretion. Mol. Biol. Cell 18, 3764-75 (2007); Ostrovsky, O., Ahmed, N.T. & Argon, Y. The chaperone activity of GRP94 toward insulin-likegrowth factor II is necessary for the stress response to serumdeprivation. Mol. Biol. Cell 20, 1855-64 (2009)). C2C12 cells weremaintained and cultured in DMEM in the presence of 10% FBS and 1%penicillin/streptomycin (Culture medium). C2C12 is an immortal line ofmouse skeletal myoblasts originally derived from satellite cells fromthe thigh muscle of a two month old female mouse donor. These cellsdifferentiate well into myocytes under appropriate culture conditions.Here, cells were induced to differentiate by replacing the culturemedium with DMEM supplemented with 2% horse serum and 1%penicillin/streptomycin (Differentiation medium) for 36-48 hours.Secreted IGF-II was quantified by using IGF-II mouse ELISA kit (Abcam,AB100696) according to the manufacturer's instructions. Briefly, aftershifting the culture medium to differentiation medium, C2C12 cells weretreated for 24 hrs with the indicated compounds. Media from eachexperimental condition was then transferred into ELISA plates coatedwith anti-IGF-II and incubated overnight at 4° C. The bound IGF-II wasdetected with a biotinylated anti-IGF-II antibody. After the sequentialincubation with HRP conjugated streptavidin, TMB One-step substratereagent and the Stop solution, the absorbance was measured at 450 nm.The secreted IGF-II was quantified against a standard curve generatedwith recombinant IGF-II provided by the kit.

TLR9-Trafficking Assay

(Yang, Y. et al. Heat shock protein gp96 is a master chaperone fortoll-like receptors and is important in the innate function ofmacrophages. Immunity 26, 215-26 (2007)). HEK 293T cells weretransfected with pUNO-hTLR9-HA (Invivogen) using X-tremgene HP (Roche)according to the manufacturer's instructions. At 24 hrspost-transfection, cells were split onto cell culture chamber slides(Lab-Tek). Cells were then treated for 24 hrs with indicated compound atvarying concentrations. After treatment, cells were fixed for 20 min in4% paraformaldehyde in PBS, permeabilized with 0.1% Triton-X 100 in PBSfor 10 min, blocked with 3% BSA in PBS for 30 min, followed by stainingfor 1 hr with an anti-HA antibody (Abcam, ab9110) or a normal rabbitIgG. Cells were washed with PBS, stained with an anti-rabbit-Cy3antibody (Invitrogen) and finally mounted in the dark at 4° C. withProlong Gold Antifade reagent (with DAPI). Cells were visualized under aconfocal microscope (Leica Upright Confocal SP5). Fluorescence intensitywas quantified using MetaMorph Microscopy Automation and Image AnalysisSoftware (Molecular Devices Inc.) and normalized to the cell number.

Preparation of Crude Plasma Membranes

(Sokolowska, I. et al. Proteomic analysis of plasma membranes isolatedfrom undifferentiated and differentiated HepaRG cells. Proteome Sci. 10,47 (2012)). All the steps were performed at 4° C. and all the bufferswere chilled on ice before use. The cells were gently scraped in PBS,pelleted by centrifugation at 600×g for 5 min and resuspended in 1 mL 1×Hypotonic Extraction Buffer (Sigma, H8412, 10 mM HEPES, pH7.8, 1 mMEGTA, 25 mM KCl) for 20 min to allow the cells to swell. Then, cellswere collected at 1000×g for 5 min, resuspended in 0.5 mL 1× IsotonicExtraction Buffer (Sigma, 13533, 10 mM HEPES, pH 7.8, 0.25M sucrose, 1mM EGTA, 25 mM KCl), homogenized with 20 strokes of the Douncehomogenizer and then centrifuged for 10 min at 1000 g. The supernatantwith the floating lipid layer was carefully collected and layered on topof 12 mL of 30% Percoll (Sigma, P4937) in Isotonic Extraction Buffer,followed by ultracentrifugation at 28,184 rpm in a TH641 rotor (ThermoScientific) for 45 min. The crude plasma membrane fraction was visibleas a ring at 5.4 cm from the bottom of the tube.

Angiogenesis Studies with HUVEC Cells Purified from the Plasma ofDiabetic Patients:

HUVECs were isolated from freshly collected umbilical veins bycollagenase treatment (Jaffe et al., 1973) applied to at least threedifferent cords. Cells were maintained in endothelial basal medium (EBM)supplemented with 10% (v/v) FBS, 100 units/ml penicillin, 10 μg/mlstreptomycin, 0.1% (v/v) rHEGF, 01% (v/v) hydrocortisone and 0.4% bovinebrain extract, at 37° C. in a humidified 95% air, 5% CO₂ atmosphere,until the cells reached sub-confluence. HUVECs at the 4^(th)-5^(th)passage were seeded at the density of 25×10⁴/well in 12-well (2 ml each)plates in EBM supplemented with 10% FBS, and allowed to attach to wellplastics for 24 h. A fresh aliquot (2 ml) of serum-free medium was addedwith native Grp94 (10 and 100 ng/ml, final concentrations), peak 2 fromthe mono-Q column (10 ng/ml), both with and without the inhibitors intriplicate wells. The inhibitors were added to cells immediately beforethe addition of Grp94 and peak 2. Wells with inhibitors alone and withthe diluent (DMSO) in which inhibitors were dissolved served ascontrols. After 18 h-incubation, morphologic examination of cells wasperformed with the Leica DMI 4000B microscope. The medium was thencollected and cells washed with PBS, detached from the wells with asolution of 0.05% trypsin and 0.2% EDTA and counted in a hemocytometer.PU-H54 was used at the final concentrations of 1 and 10 μM. Pictures ofHUVECs are representative and show overlapping features for eachcondition.

TNFα ELISA Protocol:

Mouse macrophage cells, RAW264.7, were cultured in DMEM medium(Invitrogen) with 2 mM Glutamax, 50 U/mL penicillin/streptomycin, 10 mMHEPES, 1 mM sodium pyruvate and 10% low endotoxin FBS. The cells werekept in a humidified cell incubator with CO2 at 37° C.Lipopolysaccharides (LPS, Sigma) and CpG DNA ODN1585(5′-G*G*GGTCAACGTTGAGG*G*G*G*G-3′ (SEQ ID NO: 5), IDT) were used tostimulate TNF-α production in RAW264.7 cells. TNFα production wasdetermined using the mouse TNF-α ELISA MAX Set (Biolegend) fromsupernatants of cells pretreated with inhibitors. Briefly, RAW264.7cells were pretreated for 2 hours with the indicated concentrations ofthe inhibitors, then stimulated with 10 ng/mL LPS or 2.5 uM CpG DNA for18 hours. Media from each experimental condition was then transferredinto ELISA plates (pre-coated with Capture Antibody and blocked) andincubated at room temperature (RT) for 2 hour with shaking. The capturedTNF-α was detected with Detection Antibody in the kit. After thesequential incubation with HRP conjugated streptavidin, TMB substratereagent and the Stop solution, the absorbance was measured at 450 nm.The produced TNF-α was finally quantified against a standard curvegenerated with recombinant TNF-α provided by the kit.

6.2 Preparation of Grp94 Inhibitors

6.2.1 Synthesis of Compounds of Formula 4a-t (Scheme 1)

General Procedure for Synthesis of 8-aryl Sulfanyl Derivatives 2a-w

Method A: Conventional Heating Reaction.

8-Mercaptoadenine (3.6 mmol), neocuproine hydrate (0.36 mmol), CuI (0.36mmol), NaO-t-Bu (7.2 mmol), respective aryl iodide (10.8 mmol), andanhydrous DMF (24 mL) were taken in a round bottom flask flushed withnitrogen. The flask was sealed with Teflon tape, heated at 110° C., andmagnetically stirred for 24-36 h under nitrogen. Solvent was removedunder reduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5).

Method B: Microwave Coupling Reaction.

In a conical-bottomed microwave vial, the mixture of 8 mercaptoadenine(0.1 mmol), respective aryl iodide (0.1 mmol), CuI (0.02 mmol), NaOt-Bu(0.3 mmol) and t-butyl ammonium bromide (0.02 mmol) in DMF (2 mL) wascharged. The sealed vial was irradiated in the microwave for 1.5 h at150° C. After cooling, the reaction mixture was condensed under reducedpressure and purified by flash chromatography (CH₂Cl₂:MeOH:AcOH,20:1:0.5).

8-((4-Bromo-2-ethylphenyl)thio)-9H-purin-6-amine (2a)

Obtained by method B as a light yellow solid in 49% yield. MS (ESI): m/z351.8 [M+H]⁺.

8-((4-Bromo-2-chlorophenyl)thio)-9H-purin-6-amine (2b)

Obtained by method B as a light yellow solid in 42% yield. MS (ESI): m/z357.6 [M+H]⁺.

8-((4-Chloro-2-(trifluoromethyl)phenyl)thio)-9H-purin-6-amine (2c)

Obtained by method B as a light yellow solid in 43% yield. MS (ESI): m/z343.9 [M−H]⁻.

8-((2,4-Bis(trifluoromethyl)phenyl)thio)-9H-purin-6-amine (2d)

Obtained by method B as a light yellow solid in 45% yield. MS (ESI): m/z380.0 [M+H]⁺.

8-((3-Bromo-5-chlorophenyl)thio)-9H-purin-6-amine (2e)

Obtained by method B as a light yellow solid in 42% yield. MS (ESI): m/z358.1 [M+H]⁺.

8-((3,5-Dibromophenyl)thio)-9H-purin-6-amine (2f)

Obtained by method B as a light yellow solid in 40% yield. MS (ESI): m/z401.9 [M+H]⁺.

8-((3-Bromo-5-iodophenyl)thio)-9H-purin-6-amine (2g)

Obtained by method B as a light yellow solid in 16% yield. MS (ESI): m/z449.8 [M+H]⁺.

8-((3-Bromo-5-(trifluoromethoxy)phenyl)thio)-9H-purin-6-amine (2h)

Obtained by method A as a light yellow solid in 41% yield. MS (ESI): m/z407.8 [M+H]⁺.

8-((2,3-Dichlorophenyl)thio)-9H-purin-6-amine (2i)

Obtained by method A as a yellow solid in 47% yield. MS (ESI): m/z 311.9[M+H]⁺.

8-((3,4-Dichlorophenyl)thio)-9H-purin-6-amine (2j)

Obtained by method B as a yellow solid in 69% yield. MS (ESI): m/z 312.0[M+H]⁺.

8-((3,4,5-Trichlorophenyl)thio)-9H-purin-6-amine (2k)

Obtained by method A as a light yellow solid in 42% yield. MS (ESI): m/z347.8 [M+H]⁺.

8-((2,3,4-Trichlorophenyl)thio)-9H-purin-6-amine (2l)

Obtained by method A a yellow solid in 43% yield. MS (ESI): m/z 347.7[M+H]⁺.

8-((2,3,5-Trichlorophenyl)thio)-9H-purin-6-amine (2m)

Obtained by method A as a light yellow solid in 49% yield. MS (ESI): m/z347.4 [M+H]⁺.

8-((5-Bromopyridin-2-yl)thio)-9H-purin-6-amine (2n)

Obtained by method B as a light yellow solid in 40% yield. MS (ESI): m/z324.9 [M+H]⁺.

8-(Naphthalen-1-ylthio)-9H-purin-6-amine (2o)

Obtained by method A as a yellow solid in 39% yield. MS (ESI): m/z 294.0[M+H]⁺.

8-((4-Chloronaphthalen-1-yl)thio)-9H-purin-6-amine (2p)

Obtained by method B as a light yellow solid in 39% yield. MS (ESI): m/z328.4 [M+H]⁺.

8-((4,6-Dichloroquinolin-8-yl)thio)-9H-purin-6-amine (2q)

Obtained by method B as a light yellow solid in 39% yield. MS (ESI): m/z362.9 [M+H]⁺.

8-((4-(1H-Pyrrol-1-yl)phenyl)thio)-9H-purin-6-amine (2r)

Obtained by method B as a yellow solid in 58% yield. MS (ESI): m/z 309.3[M+H]⁺.

8-((5-Bromo-l-(4-methoxybenzyl)-1H-indol-7-yl)thio)-9H-purin-6-amine(2s)

Obtained by method B as a white solid in 53% yield. MS (ESI): m/z 483.3[M+H]⁺.

8-((5-Bromo-1-(4-methoxybenzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)thio)-9H-purin-6-amine(2t)

Obtained by method B as a light yellow solid in 47% yield. MS (ESI): m/z483.1 [M+H]⁺.

8-(2,4-Dimethyl-phenylsulfanyl)adenine (2u)

Obtained by method B as a white solid in 62% yield. ¹H NMR (400 MHz,DMSO) δ 13.2 (br s, 1H), 8.05 (s, 1H), 7.03-7.26 (m, 5H), 2.30 (s, 3H),2.26 (s, 3H); ¹³C NMR (100 MHz, DMSO) δ 154.6, 152.2, 139.4, 138.5,133.0, 131.6, 127.7, 20.6, 20.24; MS (ESI): m/z 272.1 [M+H]⁺.

8-((3,5-Bis(trifluoromethyl)phenyl)thio)-9H-purin-6-amine (2v)

Obtained by method A in 57% yield. ¹H NMR (600 MHz, DMSO) δ 13.6 (br s,1H), 8.09-8.14 (m, 4H), 7.44 (br s, 2H); MS (ESI): m/z 380.1 [M+H]⁺.

8-(Mesitylthio)-9H-purin-6-amine (2w)

Obtained by method A in 53% yield. ¹H NMR (400 MHz, DMSO) δ 13.1 (br s,1H), 8.02 (s, 1H), 6.93-7.04 (m, 5H), 2.33 (s, 6H), 2.25 (s, 3H); MS(ESI): m/z 286.1 [M+H]⁺.

General Procedure for Synthesis of N9 and N3 Alkylated 8-aryl SulfanylDerivatives 3a-t and 4a-t

8-Arylsulfanyl adenine (2a-r, 1.21 mmol) was dissolved in DMF (15 mL)and Cs₂CO₃ (1.45 mmol) and 5-bromo-pent-1-yne (2.42 mmol) were added andthe mixture was stirred under nitrogen at 40° C. for 2-6 h. Solvent wasremoved under reduced pressure and the resulting residue waschromatographed (CH₂Cl₂:MeOH:AcOH, 20:1:0.5) to afford desired compounds3a-t and 4a-t.

8-((4-Bromo-2-ethylphenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (3a)

Obtained as a white solid in 40% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.30(1H, s), 7.44 (1H, d, J=2.1 Hz), 7.27-7.29 (1H, m), 7.15 (1H, d, J=8.3Hz), 5.89 (2H, br s), 4.29 (2H, t, J=7.4 Hz), 2.28 (2H, td, J=6.9, 2.5Hz), 2.01-2.06 (2H, m), 1.98 (1H, t, J=2.6 Hz); ¹³C-NMR (CDCl₃) δ 154.3,152.8, 151.7, 147.3, 145.9, 134.0, 132.3, 130.2, 128.2, 123.4, 114.9,82.4, 69.5, 42.8, 28.3, 27.2 16.0, 14.5; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₈H₁₉N₅SBr, 416.0545; found 416.0536.

8-((4-Bromo-2-chlorophenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3b)

Obtained as a white solid in 39% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.29(1H, s), 7.54 (1H, d, J=2.0 Hz), 7.24 (1H, dd, J=8.4, 2.0 Hz), 6.99 (1H,d, J=8.5 Hz), 5.72 (2H, br s), 4.26 (2H, t, J=7.3 Hz), 2.20 (2H, td,J=6.9, 2.6 Hz), 1.96 (2H, p, J=7.2 Hz), 1.91 (1H, t, J=2.6 Hz); ¹³C-NMR(CDCl₃) δ 154.6, 153.5, 151.7, 143.7, 135.1, 132.9, 132.4, 130.9, 130.5,122.2, 116.5, 82.2, 69.6, 43.1, 28.4, 16.1; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₆H₁₄N₅SBrCl, 421.9842; found 421.9823.

8-((4-Chloro-2-(trifluoromethyl)phenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3c)

Obtained as a white solid in 41% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.34(1H, s), 7.72 (1H, s), 7.41 (1H, d, J=8.1 Hz), 7.33 (1H, d, J=8.3 Hz),6.23 (2H, br s), 4.29 (2H, t, J=7.1 Hz), 2.21-2.28 (2H, m), 1.95-2.02(3H, m); ¹³C-NMR (CDCl₃) δ 154.9, 151.6, 143.7, 134.5, 132.7, 131.4,131.0, 129.1, 127.7, 124.0, 121.3, 120.3, 82.1, 70.6, 43.0, 28.3, 15.9;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₄N₅SF₃Cl₂, 412.0611; found412.0612.

8-((2,4-Bis(trifluoromethyl)phenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3d)

Obtained as a white solid in 39% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.36(1H, s), 7.96 (2H, s), 7.83 (1H, s), 5.70 (2H, br s), 4.37 (2H, t, J=7.2Hz), 2.27 (2H, td, J=6.9, 2.6 Hz), 1.98-2.07 (3H, m); ¹³C-NMR (CDCl₃) δ154.6, 153.5, 151.7, 143.6, 134.3, 132.9, 132.6, 130.7, 124.1, 121.4,120.3, 82.2, 69.6, 42.9, 28.3, 16.0; MS (ESI): m/z 446.1 [M+H]⁺.

8-((3-Bromo-5-chlorophenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3e)

Obtained as a white solid in 37% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.36(1H, s), 7.42-7.44 (2H, m), 7.33 (1H, s), 6.19 (2H, br s), 4.33 (2H, t,J=7.3 Hz), 2.27 (2H, td, J=6.6, 2.4 Hz), 1.99-2.03 (3H, m); ¹³C-NMR(CDCl₃) δ 154.9, 153.5, 151.5, 143.5, 135.9, 134.7, 131.2, 131.0, 128.7,123.4, 120.3, 82.3, 69.7, 43.0, 28.3, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₆H₁₄N₅SClBr, 423.9821; found 423.9822.

8-((3,5-Dibromophenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (3f)

Obtained as a white solid in 37% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.31(1H, s), 7.62 (1H, t, J=1.6 Hz), 7.50 (2H, d, J=1.7 Hz), 6.22 (2H, brs), 4.33 (2H, t, J=7.5 Hz), 2.27 (2H, td, J=6.8, 2.6 Hz), 1.99-2.05 (3H,m); ¹³C-NMR (CDCl₃) δ 154.2, 152.9, 151.6, 145.8, 134.1, 131.8, 128.2,123.6, 114.5, 82.4, 69.7, 43.0, 28.2, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₆H₁₄N₅SBr₂, 465.9337; found 465.9329.

8-((3,5-Dibromophenyl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine (4f)

Obtained as a white solid in 16% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.04(1H, s), 7.66 (2H, s), 7.52-7.55 (1H, m), 4.22 (2H, t, J=6.2 Hz),2.18-2.24 (4H, m), 2.05-2.08 (1H, m); HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₆H₁₄N₅SBr₂, 465.9337; found 465.9331.

8-((3-Bromo-5-iodophenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (3g)

Obtained as a white solid in 35% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.29(1H, s), 7.79 (1H, s), 7.70 (1H, s), 7.53 (1H, s), 6.57 (2H, bs), 4.33(2H, t, J=7.4 Hz), 2.27 (2H, td, J=6.6, 2.4 Hz), 1.97-2.04 (3H, m);¹³C-NMR (CDCl₃) δ 154.9, 152.8, 151.4, 144.1, 139.6, 137.5, 134.5,132.5, 123.6, 119.2, 94.8, 82.2, 69.7, 43.1, 28.2, 16.0; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₆H₁₄N₅SBrI, 513.9198; found 513.9202.

8-((3-Bromo-5-(trifluoromethoxy)phenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3h, PDP-120-A)

Obtained as a yellow solid in 36% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.33(1H, s), 7.49 (1H, t, J=1.5 Hz), 7.33 (1H, s), 7.25 (1H, s), 6.15 (2H,bs), 4.34 (2H, t, J=7.4 Hz), 2.27 (2H, td, J=6.8, 2.6 Hz), 2.02-2.05(2H, m), 1.99 (1H, t, J=2.5 Hz); ¹³C-NMR (CDCl₃) δ 161.0, 154.8, 153.2,151.5, 149.8, 143.6, 135.1, 131.1, 123.9, 123.5, 121.4, 120.2, 82.2,69.6, 43.0, 28.3, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₄N₅OF₃SBr,474.0034; found 474.0035.

8-((3-Bromo-5-(trifluoromethoxy)phenyl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine(4h)

Obtained as a yellow solid in 14% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.05(1H, s), 7.63 (1H, s), 7.41 (1H, s), 7.23 (1H, s), 4.50 (2H, t, J=7.4Hz), 2.20-2.23 (2H, m), 2.04-2.07 (3H, m); ¹³C-NMR (CDCl₃) δ 158.6,153.1, 152.0, 149.3, 145.2, 142.5, 138.1, 135.8, 130.9, 122.5, 121.3,120.1, 81.7, 70.7, 53.5, 26.8, 15.3; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₁₄N₅OF₃SBr, 474.0034; found 474.0033.

8-((3,4-Dichlorophenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (3i)

Obtained as a yellow solid in 54% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.25(1H, s), 7.58 (1H, d, J=2.2 Hz), 7.40 (1H, d, J=8.4 Hz), 7.25 (1H, dd,J=8.3, 2.1 Hz), 6.97 (2H, br s), 4.32 (2H, t, J=7.4 Hz), 2.28 (2H, td,J=6.8, 2.6 Hz), 1.97-2.02 (3H, m); ¹³C-NMR (CDCl₃) δ 154.8, 153.3,151.5, 144.6, 133.6, 133.0, 132.5, 131.3, 130.6, 130.2, 120.1, 82.3,69.6, 42.9, 28.2, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₄N₅SCl₂,378.0347; found 378.0353.

8-((3,4-Dichlorophenyl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine (4i)

Obtained as a yellow solid in 18% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.07(1H, s), 7.70 (1H, d, J=2.2 Hz), 7.42-7.44 (2H, m), 4.46 (2H, t, J=6.6Hz), 2.23-2.25 (2H, m), 2.16-2.20 (2H, m), 2.08-2.10 (1H, m); ¹³C-NMR(CDCl₃) δ 159.3, 152.5, 151.1, 142.5, 133.4, 132.9, 132.5, 131.9, 131.0,130.7, 121.6, 81.7, 70.8, 49.2, 26.9, 15.3; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₆H₁₄N₅SCl₂, 378.0347; found 378.0359.

8-((2,3-Dichlorophenyl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (3j)

Obtained as a white solid in 34% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.38(1H, s), 7.38 (1H, d, J=8.0 Hz), 7.10 (1H, t, J=7.9 Hz), 6.93 (1H, d,J=8.0 Hz), 5.89 (2H, br s), 4.33 (2H, t, J=7.3 Hz), 2.25 (2H, td, J=6.8,2.6 Hz), 2.02 (2H, p, J=7.0 Hz), 1.97 (1H, t, J=2.6 Hz); ¹³C-NMR (CDCl₃)δ 154.8, 153.6, 151.6, 143.4, 134.5, 134.2, 131.5, 129.4, 128.1, 127.9,120.5, 82.2, 69.6, 43.2, 28.4, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₆H₁₄N₅SCl₂, 378.0347; found 378.0342.

9-(Pent-4-yn-1-yl)-8-((3,4,5-trichlorophenyl)thio)-9H-purin-6-amine (3k)

Obtained as a yellow solid in 39% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.35(1H, s), 7.47 (2H, s), 5.74 (2H, br s), 4.34 (2H, t, J=7.3 Hz), 2.28(2H, td, J=6.9, 2.8 Hz), 2.03-2.07 (2H, m), 2.01 (1H, t, J=2.6 Hz);¹³C-NMR (CDCl₃) δ 154.8, 153.2, 151.5, 149.8, 143.6, 135.1, 131.1,123.9, 123.5, 121.4, 120.2, 82.2, 69.6, 43.0, 28.3, 16.0; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₆H1₃N₅SCl₃, 411.9957; found 411.9944.

9-(Pent-4-yn-1-yl)-8-((2,3,5-trichlorophenyl)thio)-9H-purin-6-amine (3l)

Obtained as a yellow solid in 41% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.30(1H, s), 7.39 (1H, d, J=2.1 Hz), 6.97 (1H, d, J=2.1 Hz); 6.80 (2H, brs), 4.36 (2H, t, J=7.2 Hz), 2.27 (2H, td, J=6.8, 2.5 Hz), 2.03 (2H,pentet, J=6.9 Hz), 1.97 (1H, t, J=2.5 Hz); ¹³C-NMR (CDCl₃) δ 155.2,152.9, 151.3, 142.5, 135.4, 134.7, 133.4, 130.0, 129.3, 127.9, 120.3,82.0, 69.7, 43.3, 28.4, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₆H1₃N₅SCl₃, 411.9957; found 411.9953.

3-(Pent-4-yn-1-yl)-8-((2,3,5-trichlorophenyl)thio)-3H-purin-6-amine (4l)

Obtained as a yellow solid in 16% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.09(1H, s), 7.31 (1H, s), 7.25 (1H, s), 6.24 (2H, br s), 4.51 (2H, t, J=6.8Hz), 2.17-2.23 (2H, m), 2.06-2.09 (3H, m); ¹³C-NMR (CDCl₃) δ 156.3,153.9, 150.5, 143.4, 138.0, 133.9, 132.5, 129.8, 128.3, 127.9, 121.3,81.7, 70.7, 49.3, 26.9, 15.3; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₆H1₃N₅SCl₃, 411.9957; found 411.9969.

9-(Pent-4-yn-1-yl)-8-((2,3,4-trichlorophenyl)thio)-9H-purin-6-amine (3m)

Obtained as a yellow solid in 39% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.35(1H, s), 7.29 (1H, d, J=8.6 Hz), 6.97 (1H, d, J=8.6 Hz), 6.18 (2H, bs),4.34 (2H, t, J=7.3 Hz), 2.26 (2H, td, J=6.8, 2.4 Hz), 2.03 (2H, pentet,J=6.9 Hz), 1.98 (1H, t, J=2.5 Hz); ¹³C-NMR (CDCl₃) δ 154.8, 153.6,151.5, 143.2, 133.5, 133.4, 133.0, 132.4, 128.8, 128.4, 120.5, 82.1,69.7, 43.1, 28.4, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H1₃N₅SCl₃,411.9957; found 411.9967.

3-(Pent-4-yn-1-yl)-8-((2,3,4-trichlorophenyl)thio)-3H-purin-6-amine (4m)

Obtained as a yellow solid in 15% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.05(1H, s), 7.24-7.26 (2H, m), 6.97 (1H, d, J=8.6 Hz), 4.48 (2H, t, J=6.7Hz), 2.06-2.20 (5H, m); ¹³C-NMR (CDCl₃) δ 157.7, 153.6, 150.7, 143.0,134.5, 134.2, 132.4, 132.2, 129.7, 128.1, 121.6, 81.7, 70.7, 49.1, 27.0,15.3; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H1₃N₅SCl₃, 411.9957; found411.9963.

8-((5-Bromopyridin-2-yl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (3n)

Obtained as a white solid in 34% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.44(1H, d, J=2.2 Hz), 8.39 (1H, s), 7.71 (1H, dd, J=8.4, 2.3 Hz), 7.17 (1H,d, J=8.4 Hz), 5.85 (2H, br s), 4.36 (2H, t, J=7.3 Hz), 2.25 (2H, td,J=6.9, 2.5 Hz), 2.07 (2H, pentet, J=7.0 Hz), 1.93 (2H, t, J=2.6 Hz);¹³C-NMR (CDCl₃) δ 155.0, 154.7, 153.7, 151.6, 151.2, 142.1, 139.9,123.6, 120.7, 118.6, 82.4, 69.4, 43.3, 28.4, 16.0; HRMS (ESI) m/z [M+H]⁺calcd. for C₁₅H₁₄N₆SBr, 389.0184; found 389.0201.

8-((5-Bromopyridin-2-yl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine (4n)

Obtained as a white solid in 15% yield. ¹H-NMR (400 MHz, CDCl₃) δ 7.97(1H, s), 7.50 (1H, s), 7.27 (1H, d, J=8.4 Hz), 7.19-7.21 (1H, m), 5.72(2H, br s), 4.42 (2H, t, J=6.2 Hz), 2.15-2.18 (4H, m), 1.99-2.01 (1H,m); MS (ESI): m/z 391.1 [M+H]⁺.

8-(Naphthalen-1-ylthio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (3o)

Obtained as a white solid in 33% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.37(1H, d, J=8.1 Hz), 8.28 (1H, s), 7.85-7.87 (2H, m), 7.64 (1H, d, J=7.1Hz), 7.54-7.60 (2H, m), 7.40-7.43 (1H, m), 5.99 (2H, br s), 4.29 (2H, t,J=7.3 Hz), 2.18 (2H, td, J=6.9, 2.6 Hz), 1.97 (2H, pentet, J=7.1 Hz),1.90 (1H, t, J=2.5 Hz); ¹³C NMR (CDCl₃) δ 154.4, 152.7, 151.7, 146.5,134.3, 133.1, 131.9, 129.9, 128.8, 127.4, 127.2, 126.7, 125.9, 124.9,120.0, 82.4, 69.5, 42.9, 28.1, 16.1; MS (ESI): m/z 360.5 [M+H]⁺.

8-((4-Chloronaphthalen-1-yl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3p)

Obtained as a white solid in 35% yield. ¹H-NMR (500 MHz, CDCl₃) δ 8.41(1H, d, J=7.9 Hz), 8.31 (1H, d, J=8.2 Hz), 7.96 (1H, s), 7.87 (1H, d,J=6.8 Hz), 7.55-7.63 (3H, m), 4.40 (2H, t, J=7.3 Hz), 2.13-2.15 (2H, m),2.04-2.10 (3H, m); MS (ESI): m/z 394.2 [M+H]⁺.

8-((4,6-Dichloroquinolin-8-yl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3q)

Obtained as a white solid in 37% yield. ¹H-NMR (500 MHz, CDCl₃) δ 8.77(1H, d, J=4.8 Hz), 8.38 (1H, d, J=2.2 Hz), 8.36 (1H, s), 8.28 (1H, d,J=2.2 Hz), 7.11 (1H, J=4.8 Hz), 6.15 (2H, br s), 4.34 (2H, t, J=7.3 Hz),2.23 (2H, td, J=6.8, 2.6 Hz), 2.02 (2H, pentet, J=6.8 Hz), 1.87 (2H, t,J=2.6 Hz).

8-((4,6-Dichloroquinolin-8-yl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine(4q)

Obtained as a white solid in 12% yield. ¹H-NMR (500 MHz, CDCl₃) δ 8.75(1H, d, J=4.7 Hz), 8.30-8.31 (2H, m), 8.08 (1H, s), 7.52 (1H, J=4.7 Hz),4.50 (2H, t, J=6.3 Hz), 2.19-2.24 (4H, m), 2.07 (1H, t, J=2.4 Hz).

8-(4-(1H-Pyrrol-1-yl)phenylthio)-9-(pent-4-ynyl)-9H-purin-6-amine (3r)

Obtained as a white solid in 52% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.37(s, 1H), 7.55 (2H, d, J=8.2 Hz), 7.38 (2H, d, J=8.2 Hz), 7.07 (2H, d,J=4.3 Hz), 6.36 (2H, d, J=4.3 Hz), 5.76 (2H, br s), 4.33 (2H, t, J=7.1Hz), 2.22-2.29 (2H, m), 1.98-2.07 (3H, m); ¹³C NMR (CDCl₃) δ 154.3,152.9, 151.7, 146.3, 140.9, 133.1, 126.8, 121.1, 120.4, 119.1, 111.1,82.4, 69.5, 42.8, 28.2, 16.1; HRMS (ESI) m/z [M+H]⁺ calcd. forC₂₀H₁₉N₆S, 375.1392; found 375.1397.

8-(4-(1H-Pyrrol-1-yl)phenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (4r)

Obtained as a white solid in 19% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.02(1H, s), 7.68 (2H, d, J=8.4 Hz), 7.46 (2H, d, J=8.5 Hz), 7.06-7.08 (2H,m), 6.34 (2H, d, J=2.0 Hz), 5.72 (2H, br s), 4.47 (2H, t, J=5.6 Hz),2.19-2.26 (4H, m), 2.00-2.04 (1H, m); ¹³C NMR (CDCl₃) δ 159.5, 152.2,151.6, 142.1, 141.0, 134.8, 121.1, 119.5, 111.2, 82.2, 71.1, 54.1, 27.4,15.7; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₀H₁₉N₆S, 375.1392; found375.1395.

8-((5-Bromo-1-(4-methoxybenzyl)-1H-indol-7-yl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3s)

Obtained as a white solid in 25% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.22(1H, s), 7.86 (1H, d, J=1.7 Hz), 7.50 (1H, d, J=1.5 Hz), 7.12 (1H, d,J=3.1 Hz), 6.73 (2H, d, J=8.5 Hz), 6.61 (2H, d, J=8.6 Hz), 6.57 (1H, d,J=3.1 Hz), 5.75 (2H, s), 4.14 (2H, t, J=7.2 Hz), 3.63 (3H, s), 2.22 (2H,td, J=6.9, 2.5 Hz), 2.02-2.06 (2H, m), 1.98 (1H, t, J=2.6 Hz); ¹³C-NMR(CDCl₃) δ 158.7, 153.8, 152.2, 151.6, 147.9, 134.6, 133.9, 132.9, 132.5,129.8, 126.9, 126.2, 113.7, 113.0, 112.4, 110.3, 102.2, 82.4, 69.6,55.1, 51.4, 42.5, 28.2, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd. forC₂₆H₂₄N₆OSBr, 547.0916; found 547.0925.

8-((5-Bromo-1-(4-methoxybenzyl)-1H-indol-7-yl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine(4s)

Obtained as a white solid in 12% yield. ¹H-NMR (400 MHz, CDCl₃) δ 7.88(1H, s), 7.82 (1H, s), 7.65 (1H, s), 7.03 (1H, d, J=3.0 Hz), 6.75 (2H,d, J=8.4 Hz), 6.58 (2H, d, J=8.6 Hz), 6.52 (1H, d, J=3.1 Hz), 5.77 (2H,s), 4.31 (2H, t, J=6.8 Hz), 3.66 (3H, s), 2.05-2.17 (5H, m); MS (ESI):m/z 549.2 [M+H]⁺.

8-((5-Bromo-1-(4-methoxybenzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(3t)

Obtained as a yellow solid in 23% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.41(1H, d, J=2.1 Hz), 8.20 (1H, s), 8.12 (1H, d, J=2.1 Hz), 7.56 (1H, s),7.24 (2H, d, J=8.6 Hz), 6.86 (2H, d, J=8.6 Hz), 6.11 (2H, br s), 5.39(2H, s), 4.36 (2H, t, J=7.2 Hz), 3.79 (3H, s), 2.32 (2H, td, J=6.9, 2.7Hz), 2.06-2.09 (2H, m), 2.03 (1H, t, J=2.6 Hz); ¹³C-NMR (CDCl₃) δ 159.6,153.9, 151.8, 148.2, 146.3, 144.9, 135.5, 130.0, 129.5, 128.2, 123.5,119.4, 117.1, 114.3, 113.3, 95.0, 82.5, 69.7, 55.3, 48.0, 42.6, 28.1,16.1; MS (ESI): m/z 548.1 [M+H]⁺.

8-((5-Bromo-1-(4-methoxybenzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine(4t)

Obtained as a yellow solid in 10% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.37(1H, s), 8.10 (1H, s), 7.93 (1H, s), 7.49 (1H, s), 7.21 (2H, d, J=8.3Hz), 6.84 (2H, d, J=8.5 Hz), 5.32 (2H, s), 4.38 (2H, t, J=7.2 Hz), 3.78(3H, s), 2.06-2.15 (5H, m); ¹³C-NMR (CDCl₃) δ 159.7, 153.8, 147.9,146.6, 144.2, 134.8, 130.6, 129.4, 128.6, 127.8, 121.7, 118.3, 115.3,114.7, 112.6, 94.7, 81.8, 70.6, 55.3, 48.9, 47.8, 28.1, 15.2; MS (ESI):m/z 548.4 [M+H]⁺.

6.2.1 Synthesis of Compounds of Formula 8a-d and 9 (Scheme 2)

8-Bromo-9H-purin-6-amine (6)

Adenine (2.2 g, 16.3 mmol) was added to a solution of bromine (6.0 mL,117.7 mmol) in water (200 mL), and the resulting mixture was stirredovernight at room temperature. The solvent was evaporated to dryness,and the brominated product 6 was used further without additionalpurification. MS (ESI): m/z 213.5/215.6 [M+H]⁺.

8-Bromo-9-(pent-4-yn-1-yl)-9H-purin-6-amine (7)

A mixture of 6 (2.0 g, 9.4 mmol), Cs₂CO₃ (4.6 g, 14.1 mmol) and5-chloropent-1-yne (1.92 ml, 18.8 mmol) in DMF (25 mL) under nitrogenprotection was heated at 80° C. for 3 h. Following solvent removal, thecrude material was purified by preparatory TLC (CH₂C1₃:MeOH:AcOH,20:1:0.1) to provide 0.52 g (23%) of 7. ¹H NMR (500 MHz, CDCl₃/MeOH-d₄)δ 8.29 (s, 1H), 4.33 (t, J=7.2 Hz, 2H), 2.28-2.33 (m, 2H), 2.09 (pentet,J=7.0 Hz, 2H), 2.02 (t, J=2.6 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃/MeOH-d₄)δ 154.4, 153.1, 151.3, 127.4, 119.9, 82.4, 69.7, 43.8, 28.2, 16.1; MS(ESI): m/z 280.1/282.2 [M+H]⁺.

General Procedure for the Synthesis of 8a-d and 9.

A mixture of thiophenol or phenol (0.069 mmol) and t-BuOK (0.069 mmol)in DMF (1.5 ml), was stirred for 15 minutes at room temperature. 7(0.057 mmol) was added and the reaction mixture was allowed to stir at80° C. for 2 h. Following solvent removal, the crude material waspurified by preparatory TLC (CH₂Cl₂:MeOH, 20:1) to afford thecorresponding derivatives 8a-d and 9.

5-((6-Amino-9-(pent-4-yn-1-yl)-9H-purin-8-yl)thio)isophthalonitrile (8a;HJP-III-26)

Yield, 10.2 mg (51%). ¹H NMR (600 MHz, CDCl₃) δ 8.38 (s, 1H), 7.98 (s,2H), 7.85 (s, 1H), 4.37 (t, J=7.3 Hz, 2H), 2.27-2.30 (m, 2H), 2.05(pentet, J=6.9 Hz, 2H), 2.01-2.03 (t, 1H); ¹³C NMR (150 MHz,CDCl₃/MeOH-d₄) δ 154.7, 153.8, 151.7, 142.2, 136.9, 136.1, 134.3, 120.5,115.8, 115.2, 82.2, 69.8, 43.1, 28.3, 15.9; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₈H₁₄N₇S, 360.1031; found 360.1028.

4-((6-Amino-9-(pent-4-yn-1-yl)-9H-purin-8-yl)thio)-2-(trifluoromethyl)benzonitrile(8b; HJP-III-29)

Yield, 16.5 mg (42%). ¹H NMR (600 MHz, CDCl₃) δ 8.39 (s, 1H), 7.85 (s,1H), 7.76 (d, J=8.2 Hz, 1H), 7.59 (d, J=8.1 Hz, 1H), 5.90 (br s, 2H),4.37 (t, J=7.4 Hz, 2H), 2.26 (td, J=6.8 and 2.6 Hz, 2H), 2.02-2.07 (m,2H), 1.97 (t, J=2.6 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃/MeOH-d₄) δ 155.1,154.1, 151.8, 141.9, 140.2, 135.5, 133.9 (q, J=32.9 Hz), 132.1, 126.9(q, J=4.7 Hz), 124.7, 121.9 (q, J=272.9 Hz), 120.7, 115.1, 108.9, 82.3,69.9, 43.3, 28.6, 16.1; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₁₄F₃N₆S,403.0953; found 403.0956.

4-((6-Amino-9-(pent-4-yn-1-yl)-9H-purin-8-yl)thio)-2-bromobenzonitrile(8c; HJP-111-32)

Yield, 6.5 mg (22%). ¹H NMR (600 MHz, CDCl₃ 6) 8.39 (s, 1H), 7.68 (d,J=1.7 Hz, 1H), 7.57 (d, J=8.2 Hz, 1H), 7.33 (dd, J=8.1 and 1.7 Hz, 1H),5.74 (br s, 2H), 4.35 (t, J=7.4 Hz, 2H), 2.26 (td, J=6.9 and 2.6 Hz,2H), 2.01-2.06 (m, 2H), 1.98 (t, J=2.6 Hz, 1H); ¹³C NMR (150 MHz,CDCl₃/MeOH-d₄) δ 155.0, 154.1, 151.8, 142.2, 140.6, 134.8, 132.7, 127.8,126.3, 120.7, 116.8, 114.8, 82.4, 69.9, 43.3, 28.6, 16.2; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₇H₁₄BrN₆S, 413.0184; found 413.0192.

4-((6-Amino-9-(pent-4-yn-1-yl)-9H-purin-8-yl)thio)-2-chlorobenzonitrile(8d; HJP-111-33)

Yield, 17.8 mg (62%). ¹H NMR (600 MHz, CDCl₃) δ 8.38 (s, 1H), 7.58 (d,J=8.3 Hz, 1H), 7.49 (d, J=1.7 Hz, 1H), 7.28 (dd, J=8.3, 1.7 Hz, 1H),6.12 (br s, 2H), 4.35 (t, J=7.4 Hz, 2H), 2.26 (td, J=6.9, 2.6 Hz, 2H),2.00-2.06 (m, 2H), 1.98 (t, J=2.6 Hz, 1H); ¹³C NMR (150 MHz,CDCl₃/MeOH-d₄) δ 155.2, 154.1, 151.7, 141.9, 140.7, 137.9, 134.5, 129.6,127.2, 120.7, 115.6, 112.3, 82.4, 69.9, 43.3, 28.6, 16.2; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₇H₁₄ClN₆S, 369.0689; found 369.0684.

8-(2,4-Dichlorophenoxy)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (9;HJP-V-45)

Yield, 16.2 mg, (51%). ¹H-NMR (600 MHz, CDCl₃) δ 8.22 (s, 1H), 7.45 (d,J=2.5 Hz, 1H), 7.43 (d, J=8.7 Hz, 1H), 7.27 (dd, J=8.7, 2.5 Hz, 1H),5.42 (br s, 2H), 4.26 (t, J=7.1 Hz, 2H), 2.27 (td, J=7.0, 2.6 Hz, 2H),2.09-2.14 (m, 2H), 1.90 (t, J=2.6 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ153.2, 152.7, 151.4, 149.9, 147.4, 132.1, 130.6, 128.9, 128.3, 127.1,123.7, 115.4, 82.5, 69.4, 41.2, 27.9, 16.1; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₆H₁₄C₁₂N₅O, 362.0575; found 362.0570.

6.2.3 Synthesis of Compounds of Formula 14a-c (Scheme 3)

General Procedure for the Synthesis of 8-arylmethyl-9H-purin-6-amines(12a-f)

In a conical-bottomed Smith process vial, the mixture of4,5,6-triaminopyrimidine (10, 0.21 g, 1.7 mmol), aryl acetic acid 11a-f(0.25 g, 1.4 mmol), and triphenyl phosphite (0.52 g, 443 μL, 1.7 mmol)in 1.5 mL anhydrous pyridine were charged. The sealed vial wasirradiated in the microwave for 30 min at 220° C. After cooling, thereaction mixture was concentrated under vacuum and the residue purifiedby column chromatography (CH₂Cl₂:MeOH, 10:0 to 10:1) to give the desiredproduct 12a-f.

8-(2,4,6-Trimethylbenzyl)-9H-purin-6-amine (12a; HJP-V-32)

Yield, 0.24 g (65%). MS (ESI) m/z 268.17 [M+H]⁺.

8-(2,4-Dichlorobenzyl)-9H-purin-6-amine (12b; HJP-V-33)

Yield, 0.33 g (79%). MS (ESI): m/z 294.04 [M+H]⁺.

8-(2,6-Dichlorobenzyl)-9H-purin-6-amine (12c; HJP-V-34)

Yield, 0.18 g (44%). MS (ESI): m/z 294.04 [M+H]⁺.

8-(3,5-Dichlorobenzyl)-9H-purin-6-amine (12d; HJP-V-35)

Yield, 0.21 g (51%). MS (ESI): m/z 294.04 [M+H]⁺.

8-(2,5-Dichlorobenzyl)-9H-purin-6-amine (12e; HJP-V-50)

Yield, 0.18 g (44%). MS (ESI): m/z 294.03 [M+H]⁺.

8-(2,3-Dichlorobenzyl)-9H-purin-6-amine (12f; HJP-V-51)

Yield, 0.24 g (58%). MS (ESI): m/z 294.04 [M+H]⁺.

General Procedure for the Synthesis of 13a-f

A mixture of 8-benzyladenine 12a-f (100 mmol), Cs₂CO₃ (100 mmol) and1-chloro-pent-4-yne (120 mmol) in DMF (1.3 mL) under nitrogen protectionwas heated at 80° C. for 1-2 h. Following solvent removal, the crudematerial was purified by preparatory TLC (CH₂Cl₂: CH₃OH—NH₃ (7N), 20:1or CH₂Cl₂:MeOH:AcOH, 15:1:0.1) to provide the corresponding9-alkyl-8-benzyladenine derivatives 13a-f.

9-(Pent-4-yn-1-yl)-8-(2,4,6-trimethylbenzyl)-9H-purin-6-amine (13a;HJP-V-36)

Yield, 12.2 mg (49%). ¹H-NMR (600 MHz, CDCl₃) δ 8.16 (s, 1H), 6.84 (s,2H), 6.09 (br s, 2H), 4.23 (t, J=7.3 Hz, 2H), 2.18-2.20 (m, 5H), 2.15(s, 6H), 1.95-1.98 (m, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 153.3, 150.2,150.1, 149.7, 136.1, 135.8, 128.2, 128.1, 117.4, 81.6, 68.8, 40.7, 27.2,27.1, 19.9, 19.3, 14.8; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₀H₂₄N₅,334.2032; found 334.2020.

8-(2,4-Dichlorobenzyl)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (13b;HJP-V-37L)

Yield, 3 mg (13%). ¹H-NMR (600 MHz, CDCl₃) δ 8.25 (s, 1H), 7.38 (d,J=2.2 Hz, 1H), 7.13 (dd, J=8.3 and 2.2 Hz, 1H), 7.01 (d, J=8.3 Hz, 1H),5.86 (br s, 2H), 4.29 (s, 2H), 4.15 (t, J=7.4 Hz, 2H), 2.17 (td, J=6.8and 2.6 Hz, 2H), 1.90-1.96 (m, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 154.3,151.7, 151.4, 150.2, 134.7, 134.1, 132.4, 131.4, 129.8, 127.8, 118.9,82.5, 70.1, 42.3, 31.3, 28.4, 15.9; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₁₆Cl₂N₅, 360.0783; found 360.0772.

8-(2,6-Dichlorobenzyl)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (13c;HJP-V-38)

Yield, 11.9 mg (48%). ¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.17 (s, 1H),7.36 (d, J=8.1 Hz, 1H), 7.21-7.24 (m, 2H), 4.47 (s, 2H), 4.35 (t, J=7.2Hz, 2H), 2.28 (td, J=6.7 and 2.5 Hz, 2H), 2.05-2.11 (m, 2H), 2.02 (t,J=2.6 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃/MeOD) δ 153.2, 150.5, 149.7,147.9, 135.2, 130.7, 128.4, 127.4, 116.9, 81.5, 69.1, 40.8, 28.9, 27.2,14.8; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₆Cl₂N₅, 360.0783; found360.0776.

8-(3,5-Dichlorobenzyl)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (13d;HJP-V-39L)

Yield, 4.1 mg (17%). ¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.22 (s, 1H),7.26 (s, 1H), 7.11 (s, 1H), 4.19 (s, 2H), 4.17 (t, J=6.9 Hz, 2H), 2.20(td, J=6.4 and 2.1 Hz, 2H), 2.05 (t, J=2.5 Hz, 1H), 1.87-1.95 (m, 2H);¹³C NMR (150 MHz, CDCl₃/MeOH-d₄) δ 151.7, 149.8, 148.6, 147.9, 136.9,134.6, 126.9, 126.3, 116.9, 81.3, 69.3, 41.3, 28.7, 26.9, 14.7; HRMS(ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₆Cl₂N₅, 360.0783; found 360.0767.

8-(2,5-Dichlorobenzyl)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (13e;HJP-V-54L)

Yield, 5 mg (21%). ¹H-NMR (600 MHz, CDCl₃) δ 8.27 (s, 1H), 7.30 (d,J=8.5 Hz, 1H), 7.17 (dd, J=8.6, 2.4 Hz, 1H), 7.07 (d, J=2.4 Hz, 1H),5.88 (br s, 2H), 4.30 (s, 2H), 4.17 (t, J=7.3 Hz, 2H), 2.17 (td, J=6.7,2.6 Hz, 2H), 1.93-1.96 (m, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 154.1, 151.3,151.2, 149.8, 135.2, 133.2, 132.1, 130.8, 130.4, 128.9, 118.8, 82.3,70.0, 42.1, 31.5, 28.2, 15.8; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₁₆Cl₂N₅, 360.0783; found 360.0776.

8-(2,3-Dichlorobenzyl)-9-(pent-4-yn-1-yl)-9H-purin-6-amine (13f;HJP-V-55)

Yield, 8.1 mg (34%). ¹H-NMR (600 MHz, CDCl₃) δ 8.26 (s, 1H), 7.34 (d,J=8.0 Hz, 1H), 7.08 (t, J=7.9, 1H), 6.92 (d, J=7.8 Hz, 1H), 5.87 (br s,2H), 4.38 (s, 2H), 4.14 (t, J=7.3 Hz, 2H), 2.16 (td, J=6.7, 2.6 Hz, 2H),1.95 (t, J=2.6 Hz, 1H), 1.89-1.94 (m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ154.2, 151.5, 151.2, 150.0, 135.9, 133.6, 132.2, 129.6, 128.3, 127.6,118.8, 82.2, 70.0, 42.1, 32.5, 28.1, 15.8; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₇H₁₆Cl₂N₅, 360.0783; found 360.0766.

General Procedure for the Synthesis of 14a-c

A mixture of 13b or 13d or 13e (100 mmol), and Cs₂CO₃ (200 mmol) in DMF(1.3 mL) was heated at 80° C. for 3 h. Following solvent removal, thecrude material was purified by preparatory TLC (CH₂Cl₂: CH₃OH—NH₃ (7N),20:1) to provide the corresponding arylketone derivatives 14a-c.

(6-Amino-9-(pent-4-yn-1-yl)-9H-purin-8-yl)(2,4-dichlorophenyl)methanone(14a; HJP-V-37T)

Yield, 12 mg (50%). ¹H-NMR (600 MHz, CDCl₃) δ 8.25 (s, 1H), 7.49 (d,J=8.2 Hz, 1H), 7.43 (d, J=1.9 Hz, 1H), 7.33 (dd, J=8.3, 1.9 Hz, 1H),6.26 (br s, 2H), 4.73 (t, J=7.2 Hz, 2H), 2.28 (td, J=7.0, 2.6 Hz, 2H),2.07-2.13 (m, 2H), 1.88 (t, J=2.6 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ185.5, 155.9, 153.5, 151.3, 144.4, 137.9, 135.5, 133.5, 131.4, 130.3,127.1, 119.6, 82.4, 69.3, 44.1, 28.9, 16.1; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₇H₁₄Cl₂N₅O, 374.0575; found 374.0571.

(6-Amino-9-(pent-4-yn-1-yl)-9H-purin-8-yl)(3,5-dichlorophenyl)methanone(14b; HJP-V-39T)

Yield, 10 mg (42%). ¹H-NMR (600 MHz, CDCl₃) δ 8.41 (s, 1H), 8.13 (d,J=1.9 Hz, 2H), 7.55 (t, J=1.9 Hz, 1H), 6.13 (br s, 2H), 4.68 (t, J=7.2Hz, 2H), 2.26 (td, J=6.9 and 2.6 Hz, 2H), 2.05-2.10 (m, 2H), 1.87 (t,J=2.6 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 181.2, 155.6, 153.7, 150.3,142.6, 137.6, 134.2, 132.2, 128.4, 118.4, 81.4, 68.2, 43.2, 27.8, 15.1;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₄Cl₂N₅O, 374.0575; found 374.0567.

(6-Amino-9-(pent-4-yn-1-yl)-9H-purin-8-yl)(2,5-dichlorophenyl)methanone(14c; HJP-V-54T)

Yield, 14 mg (58%). ¹H-NMR (600 MHz, CDCl₃) δ 8.37 (s, 1H), 7.49 (d,J=2.5 Hz, 1H), 7.31-7.33 (m, 2H), 6.14 (br s, 2H), 4.72 (t, J=7.2 Hz,2H), 2.28 (td, J=7.0 and 2.6 Hz, 2H), 2.07-2.13 (m, 2H), 1.90 (t, J=2.6Hz, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 184.1, 156.0, 154.4, 150.4, 142.6,137.6, 131.6, 130.8, 130.2, 129.5, 129.0, 118.9, 81.5, 68.2, 42.9, 27.9,15.1; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₄Cl₂N₅O, 374.0575; found374.0560.

6.2.4 Synthesis of Compounds of Formula 17a-I (Scheme 4)

8-((3,5-Dichlorophenyl)thio)-9H-purin-6-amine (15)

8-Mercaptoadenine (3.6 mmol), neocuproine hydrate (0.36 mmol), CuI (0.36mmol), NaO-t-Bu (7.2 mmol), 3,5-dichloro-iodobenzene (10.8 mmol), andanhydrous DMF (24 mL) were added to a round bottom flask flushed withnitrogen. The flask was sealed with Teflon tape and heated at 110° C.with stirring for 24-36 h under nitrogen. Solvent was removed underreduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5) to yield 15 as a yellow solid in 44% yield.¹H-NMR (400 MHz, DMSO-d₆) δ 13.49 (1H, br s), 8.13 (1H, s), 7.59 (1H,s), 7.47 (2H, s), 7.36 (2H, br s); MS (ESI): m/z 312.1 [M+H+].

General Procedure for Synthesis of N9 and N3 Alkylated 8-aryl SulfanylDerivatives 16a-1 and 17a-1

15 (1.21 mmol) was dissolved in DMF (15 mL). Cs₂CO₃ (1.45 mmol) andrespective bromides (2.42 mmol) were added and the mixture was stirredunder nitrogen at rt-60° C. for 2-6 h. Solvent was removed under reducedpressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5) to afford desired compounds 16a-1 and17a-1.

8-((3,5-Dichlorophenyl)thio)-9-(3,3,3-trifluoropropyl)-9H-purin-6-amine(16a, PDP-I-13-A)

Obtained as a white solid in 43% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.33(1H, s), 7.29 (3H, s), 6.44 (2H, br s), 4.49 (2H, t, J=6.8 Hz),2.64-2.68 (2H, m); ¹³C-NMR (CDCl₃) δ 155.0, 153.4, 151.2, 143.5, 136.0,133.8, 128.8, 128.4, 126.6, 120.2, 37.3, 33.3; HRMS (ESI): m/z [M+H]⁺calcd. for C₁₄H₁₁N₅SCl₂F3, 408.0064; found 408.0074.

8-((3,5-Dichlorophenyl)thio)-3-(3,3,3-trifluoropropyl)-3H-purin-6-amine(17a, PDP-13B)

Obtained as a white solid in 20% yield. ¹H-NMR (400 MHz, CDCl₃) δ 7.99(1H, s), 7.46 (2H, s), 7.32 (1H, s), 4.55 (2H, t, J=6.3 Hz), 2.86-2.93(2H, m); ¹³C-NMR (CDCl₃) δ 159.3, 153.1, 150.3, 142.5, 135.9, 135.2,129.7, 127.8, 125.3, 121.7, 32.7, 29.6; HRMS (ESI): m/z [M+H]⁺ calcd.for C₁₄H₁₁N₅SCl₂F3, 408.0064; found 408.0065.

8-((3,5-Dichlorophenyl)thio)-9-(4,4,4-trifluorobutyl)-9H-purin-6-amine(16b, PDP-I-15-A)

Obtained as a white solid in 44% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.36(1H, s), 7.28 (3H, s), 6.16 (2H, br s), 4.30 (2H, t, J=6.6 Hz),2.10-2.14 (2H, m), 2.03-2.05 (2H, m); ¹³C-NMR (CDCl₃) δ 154.9, 153.7,151.5, 143.2, 135.9, 134.2, 128.6, 128.1, 125.1, 120.3, 42.5, 31.3,22.5; HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₅H₁₃N₅SCl₂F3, 422.0221; found422.0222.

8-((3,5-Dichlorophenyl)thio)-3-(pent-4-yn-1-yl)-3H-purin-6-amine (17b,PDP-I-15-B)

Obtained as a white solid in 15% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.02(1H, s), 7.12 (3H, s), 5.89 (2H, s), 4.08 (2H, t, J=6.6 Hz), 2.09-2.11(4H, m); ¹³C-NMR (CDCl₃) δ 155.5, 153.1, 150.7, 149.9, 136.1, 134.2,128.0, 127.1, 125.3, 115.6, 40.1, 31.4, 21.9; MS (ESI): m/z 421.9[M+H]⁺.

8-((3,5-Dichlorophenyl)thio)-9-(5,5,5-trifluoropentyl)-9H-purin-6-amine(16c, PDP-109A)

Obtained as a white solid in 45% yield. ¹H-NMR (400 MHz, CDCl₃, δ) 8.28(1H, s), 7.28 (3H, s), 6.82 (2H, br s), 4.25 (2H, t, J=7.2 Hz),2.07-2.14 (2H, m), 1.85 (2H, pentet, J=7.4 Hz), 1.57 (2H, pentet, J=7.5Hz); ¹³C-NMR (CDCl₃) δ 155.1, 152.7, 151.2, 143.5, 135.9, 134.1, 128.6,128.2, 125.4, 120.1, 43.3, 33.1, 28.8, 19.2; HRMS (ESI): m/z [M+H]⁺calcd. for C₁₆H₁₅N₅SF₃Cl₂, 436.0377; found 436.0363.

8-((3,5-Dichlorophenyl)thio)-3-(5,5,5-trifluoropentyl)-3H-purin-6-amine(17c, PDP-109B)

Obtained as a white solid in 16% yield. ¹H-NMR (400 MHz, CDCl₃) δ 7.96(1H, s), 7.43 (2H, s), 7.17 (1H, s), 4.36 (2H, t, J=6.8 Hz), 2.05-2.17(4H, m), 1.59-1.67 (2H, m); ¹³C-NMR (CDCl₃) δ 157.8, 152.9, 150.8,142.1, 136.2, 135.1, 129.6, 127.7, 122.0, 117.7, 48.8, 33.1, 28.4, 19.0;HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₆H₁₅N₅F₃SCl₂, 436.0377; found436.0398.

8-((3,5-Dichlorophenyl)thio)-9-(6,6,6-trifluorohexyl)-9H-purin-6-amine(16d, PDP-101B)

Obtained as a white solid in 47% yield. ¹H-NMR (400 MHz, CDCl₃, δ) 8.35(1H, s), 7.29 (3H, s), 6.33 (2H, br s), 4.24 (2H, t, J=7.2 Hz),2.00-2.07 (2H, m), 1.79 (2H, pentet, J=7.4 Hz), 1.57 (2H, pentet, J=7.6Hz), 1.37 (2H, pentet, J=7.8 Hz); ¹³C-NMR (CDCl₃) δ 155.1, 152.8, 151.3,143.4, 135.9, 134.4, 128.5, 128.1, 125.8, 120.0, 43.6, 33.9, 29.4, 25.7,21.4; HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₇H₁₇N₅SCl₂F3, 450.0534; found450.0549.

8-((3,5-Dichlorophenyl)thio)-3-(6,6,6-trifluorohexyl)-3H-purin-6-amine(17d, PDP-101-A)

Obtained as a white solid in 14% yield. ¹H-NMR (500 MHz, CDCl₃) δ 7.95(1H, s), 7.43 (2H, s), 7.09 (1H, s), 4.34 (2H, t, J=7.0 Hz), 1.40-1.72(8H, m); ¹³C-NMR (CDCl₃) δ 155.0, 153.5, 151.4, 143.1, 135.9, 134.7,128.3, 127.8, 125.6, 120.3, 43.6, 33.6, 29.4, 25.7, 21.5; HRMS (ESI):m/z [M+H]⁺ calcd. for C₁₇H1₇N₅SCl₂F3, 450.0534; found 450.0539.

9-(4-Bromopentyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (16e,PDP-II-99A)

Obtained as a white solid in 43% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.36(1H, s), 7.28 (1H, s), 7.24 (2H, s), 6.08 (2H, br s), 4.27 (2H, t, J=7.2Hz), 4.09 (1H, sextet, J=6.6 Hz), 2.01-2.05 (1H, m), 1.89-1.94 (1H, m),1.72-1.78 (2H, m), 1.65 (3H, d, J=6.7 Hz); ¹³C-NMR (CDCl₃, δ) 154.9,153.6, 151.5, 143.3, 135.9, 134.7, 128.4, 127.9, 120.3, 50.1, 43.1,37.7, 28.2, 26.4; HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₆H₁₄N₅SOCl₂,461.9745; found 461.9748.

3-(4-Bromopentyl)-8-((3,5-dichlorophenyl)thio)-3H-purin-6-amine (17e,PDP-99-B)

Obtained as a white solid in 16% yield. ¹H-NMR (400 MHz, CDCl₃) δ 7.99(1H, s), 7.40 (2H, s), 7.16 (1H, s), 4.39 (2H, t, J=7.1 Hz), 4.12 (1H,sextet, J=6.5 Hz), 2.15-2.23 (2H, m), 1.80-1.86 (2H, m), 1.68 (3H, d,J=6.5 Hz); ¹³C-NMR (CDCl₃, δ) 152.5, 150.7, 148.4, 142.0, 135.9, 134.9,127.9, 126.9, 117.3, 49.9, 40.3, 37.5, 27.9, 26.4; HRMS (ESI): m/z[M+H]⁺ calcd. for C₁₆H₁₄N₅SOCl₂, 461.9745; found 461.9728.

9-(But-2-yn-1-yl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (16f,PDP-102-A)

Obtained as a white solid in 40% yield. ¹H-NMR (500 MHz, CDCl₃) δ 8.33(1H, s), 7.32 (2H, s), 7.28 (1H, s), 6.63 (2H, br s), 4.98-4.99 (2H, m),1.70 (3H, m); ¹³C-NMR (CDCl₃) δ 155.1, 153.2, 150.8, 143.3, 135.7,134.5, 128.5, 128.3, 120.0, 82.1, 71.7, 33.4, 3.5; HRMS (ESI): m/z[M+H]⁺ calcd. for C₁₅H₁₂N₅SCl₂, 364.0190; found 364.0197.

3-(But-2-yn-1-yl)-8-((3,5-dichlorophenyl)thio)-3H-purin-6-amine (17f,PDP-102B)

Obtained as a white solid in 17% yield. ¹H-NMR (500 MHz, CDCl₃) δ 8.36(1H, s), 7.40 (2H, s), 7.20 (1H, s), 5.09-5.10 (2H, m), 1.93 (3H, m);¹³C-NMR (CDCl₃) δ 158.1, 153.4, 150.5, 141.7, 137.1, 135.0, 128.5,127.1, 121.4, 86.5, 69.4, 39.7, 3.7; HRMS (ESI): m/z [M+H]⁺ calcd. forC₁₅H1₂N₅SCl₂, 364.0190; found 364.0190.

9-(But-3-yn-1-yl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (16g,PDP-I-14-A)

Obtained as a white solid in 47% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.37(1H, s), 7.28 (3H, s), 6.04 (2H, bs), 4.45 (2H, t, J=7.0 Hz), 2.76 (2H,td, J=6.8, 2.3 Hz), 1.95 (1H, t, J=2.4 Hz); ¹³C-NMR (CDCl₃) δ 154.9,153.6, 151.3, 143.7, 135.8, 134.9, 128.3, 127.9, 120.4, 79.4, 71.5,42.3, 19.5; HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₅H1₂N₅SCl₂, 364.0190;found 364.0194.

3-(But-3-yn-1-yl)-8-((3,5-dichlorophenyl)thio)-3H-purin-6-amine (17g,PDP-I-14-B)

Obtained as a white solid in 18% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.08(1H, s), 7.43 (2H, s), 7.30 (1H, s), 4.44 (2H, t, J=6.0 Hz), 2.08-2.10(3H, m); ¹³C-NMR (CDCl₃) δ 153.1, 150.3, 142.9, 136.6, 136.2, 135.2,129.5, 127.7, 121.9, 79.2, 72.5, 47.3, 19.2; HRMS (ESI): m/z [M+H]⁺calcd. for C₁₅H₁₂N₅SCl₂, 364.0190; found 364.0192.

8-((3,5-Dichlorophenyl)thio)-9-(hex-5-yn-1-yl)-9H-purin-6-amine (16h,PDP-112A)

Obtained as a white solid in 41% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.29(1H, s), 7.28 (3H, s), 6.71 (2H, bs), 4.26 (2H, t, J=7.2 Hz), 2.22 (2H,td, J=6.8, 2.7 Hz), 1.95 (1H, t, J=2.6 Hz), 1.87-1.93 (2H, m), 1.53 (2H,pentet, J=6.8 Hz); ¹³C-NMR (CDCl₃) δ 155.0, 151.2, 143.6, 135.9, 134.4,129.0, 128.5, 128.2, 120.0, 83.3, 69.1, 43.5, 28.8, 25.3, 17.9; HRMS(ESI): m/z [M+H]⁺ calcd. for C₁₇H₁₅N₅SCl₂, 392.0503; found 392.0493.

8-((3,5-Dichlorophenyl)thio)-3-(hex-5-yn-1-yl)-3H-purin-6-amine (17h,PDP-112-B)

Obtained as a white solid in 15% yield. ¹H-NMR (400 MHz, CDCl₃) δ 7.99(1H, s), 7.44 (2H, s), 7.20 (1H, s), 4.38 (2H, t, J=6.9 Hz), 2.27 (2H,td, J=6.8, 2.7 Hz), 2.11-2.15 (2H, m), 1.97 (1H, t, J=2.5 Hz), 1.58 (2H,pentet, J=6.8 Hz); ¹³C-NMR (CDCl₃) δ 156.3, 152.4, 143.4, 135.0, 134.9,129.0, 128.4, 127.0, 118.6, 83.2, 69.4, 50.2, 28.5, 25.1, 17.9; HRMS(ESI): m/z [M+H]⁺ calcd. for C₁₇H₁₅N₅SCl₂, 392.0503; found 392.0489.

4-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)butanenitrile(16i, PDP-93)

Obtained as a white solid in 41% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.34(1H, s), 7.31 (3H, s), 6.04 (2H, bs), 4.36 (2H, t, J=7.0 Hz), 2.42 (2H,t, J=7.1 Hz), 2.18 (2H, pentet, J=7.2 Hz); ¹³C-NMR (CDCl₃) δ 154.8,152.7, 151.5, 143.4, 136.0, 133.9, 129.2, 127.5, 120.2, 118.3, 42.4,25.6, 14.9; HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₅H1₃N₆SCl₂, 379.0299;found 379.0303.

4-(6-Amino-8-((3,5-dichlorophenyl)thio)-3H-purin-3-yl)butanenitrile(17i, PDP-II-93B)

Obtained as a white solid in 16% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.08(1H, s), 7.34 (2H, s), 7.31 (1H, s), 4.44 (2H, t, J=6.9 Hz), 2.50 (2H,t, J=7.0 Hz), 2.36 (2H, pentet, J=6.9 Hz); ¹³C-NMR (CDCl₃) δ 155.1,152.6, 151.3, 142.6, 136.0, 135.2, 129.5, 127.7, 120.3, 118.2, 39.6,24.8, 14.3; HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₅H1₃N₆SCl₂, 379.0299;found 379.0290.

9-(Cyclohexylmethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (16j,PDP-110-A)

Obtained as a white solid in 43% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.29(1H, s), 7.26 (3H, s), 6.68 (2H, br s), 4.06 (2H, d, J=7.5 Hz),1.85-1.88 (1H, m), 1.65-1.71 (4H, m), 1.51-1.54 (2H, m), 1.11-1.16 (2H,m), 1.02-1.06 (2H, m); ¹³C-NMR (CDCl₃) δ 154.9, 152.6, 151.5, 144.2,135.8, 134.6, 128.3, 122.8, 120.0, 49.9, 38.2, 30.5, 27.8, 26.1; HRMS(ESI): m/z [M+H]⁺ calcd. for C₁₈H₁₉N₅SCl₂, 408.0816; found 408.0805.

9-Benzyl-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (16k, PDP-107-A)

Obtained as a white solid in 48% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.43(1H, s), 7.23 (3H, s), 7.15-7.19 (3H, m), 7.04-7.05 (2H, m), 5.97 (2H,br s), 5.45 (2H, s); ¹³C-NMR (CDCl₃) δ 154.9, 153.9, 151.7, 143.6,135.6, 135.3, 134.5, 128.7, 128.2, 128.1, 127.9, 127.5, 120.8, 47.0;HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₈H₁₄N₅SCl₂, 402.0347; found402.0335.

3-Benzyl-8-((3,5-dichlorophenyl)thio)-3H-purin-6-amine (17k, PDP-107B)

Obtained as a white solid in 16% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.01(1H, s), 7.45 (3H, s), 7.34-7.38 (5H, m), 5.48 (2H, s); HRMS (ESI): m/z[M+H]⁺ calcd. for C₁₈H₁₄N₅SCl₂, 402.0347; found 402.0343.

8-((3,5-Dichlorophenyl)thio)-9-phenethyl-9H-purin-6-amine (161,PDP-127-A)

Obtained as a yellow solid in 45% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.35(1H, s), 7.22-7.26 (4H, m), 7.19-7.20 (2H, m), 7.04-7.06 (2H, m), 6.45(2H, br s), 4.47 (2H, t, J=7.2 Hz), 3.09 (2H, t, J=7.3 Hz); ¹³C-NMR(CDCl₃) δ 153.8, 151.5, 151.2, 145.1, 136.8, 135.8, 134.0, 128.9, 128.8,128.6, 128.5, 127.2, 120.1, 45.5, 35.7; HRMS (ESI): m/z [M+H]⁺ calcd.for C₁₉H₁₆N₅SCl₂, 416.0503; found 416.0508.

General Method for Synthesis of 16m

To a suspension of 8-((2,4-dichlorophenyl)thio)-9H-purin-6-amine (18,1.0 mmol) in CH₂Cl₂:toluene (0.5:2.5 mL) were added PPh₃ (4.0 mmol) andalcohol (2.0 mmol) under nitrogen protection. After stirring for 10 min.DBAD (6 mmol) was added and reaction mixture was stirred at rt for 2-5h. Following solvent removal, the crude material was purified bypreparative TLC (CH₂Cl₂:CH₃OH:AcOH, 20:1:0.1 or CH₂Cl₂:NH₃—CH₃OH (7N),20:1) to afford desired compounds 16m-.

8-((3,5-Dichlorophenyl)thio)-9-(pentan-2-yl)-9H-purin-6-amine (16m;HJP-V-123)

Yield, 7.8 mg (15%). ¹H NMR (600 MHz, CDCl₃+5 drops MeOD, 2 rotamers) δ8.17-8.21 (m, 1H), 7.40-7.56 (m, 3H), 4.76-4.79 (m, 0.4H), 4.65-4.69 (m,0.6H), 2.21-2.27 (m, 0.6H), 1.97-2.03 (m, 0.4H), 1.81-1.92 (m, 1H),1.60-1.63 (m, 3H), 1.03-1.28 (m, 2H), 0.87-0.89 (m, 3H); ¹³C NMR (150MHz, CDCl₃+5 drops MeOD) δ 153.3, 151.2, 150.0, 145.6, 135.9, 135.2,133.5, 130.2, 129.1, 128.1, 120.4, 54.4, 36.7, 19.8, 19.7, 13.6; HRMS(ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₈Cl₂N₅S, 382.0660; found 382.0663.

6.2.5 Synthesis of Compounds of Formula 20a-e (Scheme 5)

8-((2,4-Dichlorophenyl)thio)-9H-purin-6-amine (18)

8-Mercaptoadenine (1, 1.23 g, 7 mmol), 1-iodo-2,4-dichlorobenzene (3 g,11 mmol), neocuprine hydrate (0.3 g, 1.4 mmol), CuI (0.28 g, 1.4 mmol),NaOt-Bu (1.4 g, 14 mmol) and DMF (20 mL) were charged in a nitrogenprotected dry vessel. The reaction vessel was sealed and placed in anoil bath (110° C.) and stirred for 24 hrs. The reaction mixture was thencooled to room temperature and DMF was removed in vacuo. The crudematerial was purified by silica gel flash chromatography(CH₂Cl₂:CH₃OH:CH₃COOH, 60:1:0.5 to 20:1:0.5) to afford the 2.0 g (87%)of 18. MS (ESI): m/z 312.0 [M+H]⁺.

General Method for Synthesis of 19a-e and 20a-e

A mixture of 8-((2,4-dichlorophenyl)thio)-9H-purin-6-amine (18, 1.0mmol), Cs₂CO₃ (1.5 mmol), and arylethylbromide (3.0 mmol) in DMF (1.5mL) under nitrogen protection was stirred at room temperature for 1-2 h.Following solvent removal, the crude material was purified bypreparative TLC (CH₂Cl₂:CH₃OH:AcOH, 20:1:0.1) to afford desired N-9compounds.

8-((2,4-Dichlorophenyl)thio)-9-(2-(pyridin-2-yl)ethyl)-9H-purin-6-amine(19a; HJP-V-93-N9)

Yield, 7.8 mg (15%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.52 (d, J=4.9Hz, 1H), 8.25 (s, 1H), 7.58 (t, J=7.7 Hz, 1H), 7.49-7.51 (m, 1H), 7.30(d, J=8.4 Hz, 1H), 7.18-7.26 (m, 2H), 7.01 (d, J=7.7 Hz, 1H), 4.68 (t,J=6.9, 2H), 3.35 (t, J=7.0, 2H); ¹³C NMR (150 MHz, CDCl₃/MeOH-d₄) δ156.8, 153.4, 151.2, 150.9, 149.3, 146.3, 137.1, 136.9, 135.9, 134.5,130.3, 128.2, 127.7, 123.8, 122.3, 119.5, 43.7, 37.2; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₈H₁₅Cl₂N₆S, 417.0456; found 417.0447.

8-((2,4-Dichlorophenyl)thio)-9-(2-fluorophenethyl)-9H-purin-6-amine(19b; HJP-V-96)

Yield, 7.2 mg (27%). ¹H NMR (600 MHz, CDCl₃) δ 8.25 (s, 1H), 7.38 (d,J=2.0 Hz, 1H), 7.06-7.19 (m, 3H), 6.85-6.94 (m, 3H), 6.05 (br s, 2H),4.44 (t, J=7.1, 2H), 3.11 (t, J=7.2, 2H); ¹³C NMR (150 MHz, CDCl₃) δ161.4 (d, J=244.7 Hz), 153.4, 151.3, 151.2, 145.6, 135.7, 135.1, 133.1,131.1 (d, J=4.5 Hz), 130.2, 129.1 (d, J=8.1 Hz), 128.9, 128.1, 124.3 (d,J=3.7 Hz), 123.8 (d, J=15.9 Hz), 120.1, 115.5 (d, J=21.5 Hz), 43.9,29.5; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H₁₅Cl₂FN₅S, 434.0409; found434.0407.

9-(2-Chlorophenethyl)-8-((2,4-dichlorophenyl)thio)-9H-purin-6-amine(19c; HJP-V-97)

Yield, 5.2 mg (19%). ¹H NMR (600 MHz, CDCl₃) δ 8.32 (s, 1H), 7.46 (d,J=2.0 Hz, 1H), 7.46 (dd, J=8.0 and 2.0 Hz, 1H), 7.15-7.19 (m, 3H), 7.10(td, J=7.5 and 1.2 Hz, 1H), 6.94 (dd, J=7.6 and 1.8 Hz, 1H), 6.30 (bs,2H), 4.55 (t, J=7.0, 2H), 3.29 (t, J=7.0, 2H); ¹³C NMR (150 MHz, CDCl₃)δ 152.9, 151.3, 150.1, 146.3, 135.9, 135.2, 134.5, 134.4, 133.3, 131.1,130.3, 129.8, 128.8, 128.5, 128.1, 127.1, 119.9, 43.6, 33.6; HRMS (ESI)m/z [M+H]⁺ calcd. for C₁₉H₁₅Cl₃N₅S, 450.0114; found 450.0099.

8-((2,4-Dichlorophenyl)thio)-9-(2-(trifluoromethyl)phenethyl)-9H-purin-6-amine(19d; HJP-V-98)

Yield, 7.7 mg (26%). ¹H NMR (600 MHz, CDCl₃) δ 8.24 (s, 1H), 7.59 (d,J=7.7 Hz, 1H), 7.39 (s, 1H), 7.32 (t, J=7.6 Hz, 1H), 7.28 (t, J=7.4 Hz,1H), 7.11 (s, 2H), 6.94 (d, J=7.4 Hz, 1H), 6.41 (br s, 2H), 4.45 (t,J=7.1, 2H), 3.25 (t, J=7.2, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 153.2,151.2, 150.4, 146.1, 136.0, 135.4, 133.4, 132.1, 131.8, 131.4, 130.3,129.1 (q, J=29.7), 128.3, 128.1, 127.4, 126.4 (q, J=5.5), 124.8 (q,J=272.1), 119.9, 44.9, 32.7; HRMS (ESI) m/z [M+H]⁺ calcd. forC₂₀H₁₅Cl₂F₃N₅S, 484.0377; found 484.0367.

9-(3-(Isopropylamino)propyl)-8-((2,4,5-trichlorophenyl)thio)-9H-purin-6-amine(19e; HJP-V-103-N9)

Yield, 7.4 mg (18%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.20 (s, 1H),7.58 (s, 1H), 7.45 (s, 1H), 4.29 (t, J=6.9, 2H), 2.76 (septet, J=6.2,1H), 2.56 (t, J=6.8, 2H), 2.02 (pentet, J=6.8, 2H), 1.05 (d, J=6.4, 6H);¹³C NMR (150 MHz, CDCl₃/MeOH-d₄) δ 154.6, 152.9, 151.2, 144.1, 134.4,134.1, 133.8, 132.1, 131.6, 129.4, 119.7, 42.9, 41.4, 29.6, 29.2, 21.7;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H2₀C1₃N₆S, 445.0536; found 445.0520.

8-((2,4-Dichlorophenyl)thio)-3-(2-(pyridin-2-yl)ethyl)-3H-purin-6-amine(20a; HJP-V-93-N3)

Yield, 7.9 mg (15%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.59 (d, J=4.8Hz, 1H), 7.85 (s, 1H), 7.56 (t, J=7.6 Hz, 1H), 7.45 (s, 1H), 7.44 (d,J=6.7 Hz, 1H), 7.14-7.19 (m, 2H), 6.95 (d, J=7.7 Hz, 1H), 4.86 (t,J=6.4, 2H), 3.49 (t, J=6.4, 2H); ¹³C NMR (150 MHz, CDCl₃/MeOH-d₄) δ158.3, 156.6, 152.8, 150.9, 149.6, 143.0, 136.8, 136.6, 135.2, 133.4,132.9, 132.1, 129.6, 127.5, 124.1, 122.2, 49.4, 36.3; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₈H₁₅Cl₂N₆S, 417.0456; found 417.0443.

3-(3-(Isopropylamino)propyl)-8-((2,4,5-trichlorophenyl)thio)-3H-purin-6-amine(20e; HJP-V-103-N3)

Yield, 7.3 mg (18%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.06 (s, 1H),7.57 (s, 1H), 7.50 (s, 1H), 4.38 (t, J=6.9, 2H), 2.79 (septet, J=6.4,1H), 2.60 (t, J=6.5, 2H), 2.12 (pentet, J=6.5, 2H), 1.07 (d, J=6.3, 6H);¹³C NMR (150 MHz, CDCl₃/MeOH-d₄) δ 157.6, 153.1, 150.8, 143.4, 133.9,133.5, 132.8, 132.2, 131.4, 130.9, 121.9, 47.9, 42.7, 29.7, 28.9, 21.7;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₂₀Cl₃N₆S, 445.0536; found 445.0523.

General Method for Synthesis of 19f-h

To a suspension of 8-((2,4-dichlorophenyl)thio)-9H-purin-6-amine (18,1.0 mmol) in CH₂Cl₂:toluene (0.5:2.5 mL) were added PPh₃ (4.0 mmol) andalcohol (2.0 mmol) under nitrogen protection. After stirring for 10 min.DBAD (6 mmol) was added and reaction mixture was stirred at rt for 2-5h. Following solvent removal, the crude material was purified bypreparative TLC (CH₂Cl₂:CH₃OH:AcOH, 20:1:0.1 or CH₂Cl₂:NH₃—CH₃OH (7N),20:1) to afford desired compounds 19f-h.

8-((2,4-Dichlorophenyl)thio)-9-(pentan-2-yl)-9H-purin-6-amine (19f;HJP-V-114)

Yield, 7.8 mg (15%). ¹H NMR (600 MHz, CDCl₃) δ 8.22 (s, 1H), 7.43 (d,J=1.8 Hz, 1H), 7.13-7.20 (m, 2H), 6.14 (br s, 2H), 4.65-4.70 (m, 1H),2.17-2.23 (m, 1H), 1.80-1.86 (m, 1H), 1.54 (d, J=6.9 Hz, 3H), 1.12-1.19(m, 1H), 0.97-1.02 (m, 1H), 0.79 (t, J=7.4 Hz, 3H); ¹³C NMR (150 MHz,CDCl₃) δ 153.3, 151.2, 150.0, 145.6, 135.9, 135.2, 133.5, 130.2, 129.1,128.1, 120.4, 54.4, 36.7, 19.8, 19.7, 13.6; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₆H₁₈Cl₂N₅S, 382.0660; found 382.0663.

8-((2,4-Dichlorophenyl)thio)-9-(2-(pyridin-3-yl)ethyl)-9H-purin-6-amine(19g; HJP-V-116)

Yield, 7.8 mg (15%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.41 (s, 1H),8.31 (s, 1H), 8.24 (s, 1H), 7.40 (d, J=2.0 Hz, 1H), 7.37 (d, J=7.9 Hz,1H), 7.08-7.14 (m, 3H), 6.16 (br s, 2H), 4.41 (t, J=7.3, 2H), 3.07 (t,J=7.3, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 153.0, 151.0, 150.1, 148.9,147.3, 143.6, 135.6, 134.5, 134.2, 132.1, 131.5, 129.2, 127.7, 127.2,122.6, 119.0, 43.8, 31.9; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₁₅Cl₂N₆S,417.0456; found 417.0448.

8-((2,4-Dichlorophenyl)thio)-9-(2-(pyridin-4-yl)ethyl)-9H-purin-6-amine(19h; HJP-V-118)

Yield, 7.8 mg (15%). ¹H NMR (600 MHz, CDCl₃) δ 8.43 (s, 2H), 8.27 (s,1H), 7.41 (d, J=2.1 Hz, 1H), 7.13 (dd, J=8.5 and 2.2 Hz, 1H), 7.08 (d,J=8.5 Hz, 1H), 7.01 (d, J=5.0 Hz, 2H) 5.89 (br s, 2H), 4.42 (t, J=7.4,2H), 3.07 (t, J=7.4, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 154.3, 152.9,151.3, 149.9, 146.1, 144.3, 135.6, 135.2, 133.1, 128.9, 128.2, 124.3,120.2, 44.2, 35.1; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₁₅Cl₂N₆S,417.0456; found 417.0448.

8-((2,4-dichlorophenyl)thio)-9-(hex-5-yn-3-yl)-9H-purin-6-amine (19i;HJP-V-117)

Yield, 3.8 mg (24%). ¹H NMR (600 MHz, MeOD) δ 8.27 (s, 1H), 7.69 (d,J=2.2 Hz, 1H), 7.63 (d, J=8.5 Hz, 1H), 7.42 (d, J=8.5 and 2.3 Hz, 1H),4.75-4.79 (m, 1H), 3.24-3.28 (m, 1H), 2.89-2.94 (m, 1H), 2.35-2.41 (m,1H), 2.25 (t, J=2.6 Hz, 1H), 2.05-2.10 (m, 1H), 0.84 (t, J=7.4 Hz, 3H);¹³C NMR (150 MHz, MeOD) δ 153.3, 151.2, 150.0, 145.6, 135.9, 135.2,133.5, 130.2, 129.1, 128.1, 120.4, 54.4, 36.7, 19.8, 19.7, 13.6; HRMS(ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₆Cl₂N₅S, 392.0660; found 392.0663.

6.2.6 Synthesis of Compounds of Formula 22a-b (Scheme 6)

Reaction of 21 with m-CPBA to Result in Sulfoxide 22a and Sulfone 22b.

A mixture of 21 (20 mg, 0.053 mmol) and m-CPBA (18.2 mg, 0.106 mmol) inTHF:CH₂Cl₂ (2 mL) under nitrogen protection was stirred at roomtemperature for 30 min. Following solvent removal, the crude materialwas purified by preparative TLC (CH₂Cl₂:CH₃OH—NH₃ (7N), 20:1 to afforddesired products 22a and 22b.

8-((2,4-Dichlorophenyl)sulfinyl)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(22a; HJP-V-62M)

Yield, 3.4 mg (23%). ¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.29 (s, 1H),8.04 (d, J=8.5 Hz, 1H), 7.58 (dd, J=8.5, 2.0 Hz, 1H), 7.41 (d, J=2.0 Hz,1H), 4.57 (t, J=7.1 Hz, 2H), 2.30 (td, J=7.0, 2.6 Hz, 2H), 2.06-2.20 (m,2H), 1.98 (t, J=2.6 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃/MeOH-d₄) δ 155.1,152.6, 150.6, 139.2, 136.3, 131.9, 130.1, 128.7, 128.6, 119.1, 81.9,70.0, 43.5, 28.7, 16.0; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₄Cl₂N₅OS,394.0296; found 394.0279.

8-((2,4-Dichlorophenyl)sulfonyl)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(22b; HJP-V-62T)

Yield, 5.1 mg (34%). ¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.39 (s, 1H),8.24 (d, J=8.4 Hz, 1H), 7.48-7.51 (m, 2H), 6.24 (br s, 2H), 4.88 (t,J=7.6 Hz, 2H), 2.29 (td, J=7.0, 2.6 Hz, 2H), 2.10-2.16 (m, 2H), 1.92 (t,J=2.6 Hz, 1H); ¹³C NMR (150 MHz, CDCl₃/MeOH-d₄) δ 155.4, 153.3, 150.7,146.2, 142.2, 134.9, 134.8, 132.6, 132.0, 127.9, 119.3, 82.1, 69.6,44.4, 28.9, 16.1; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₄Cl₂N₅O₂S,410.0245; found 410.0228.

6.2.7 Synthesis of Compounds of Formula 24a-o (Scheme 7)

9-(2-Bromoethyl)-8-((2,4-dichlorophenyl)thio)-9H-purin-6-amine (23)

A mixture of 8-((2,4-dichlorophenyl)thio)-9H-purin-6-amine (18, 0.4 g,1.28 mmol), Cs₂CO₃ (0.63 g, 1.92 mmol), and 1,2-dibromopropane (1.21 g,0.55 mL, 6.43 mmol) in DMF (10 mL) under nitrogen protection was stirredat room temperature for 30 min. Following solvent removal, the crudematerial was purified by flash chromatography (CH₂Cl₂:CH₃OH:AcOH,100:1:0.5 to 20:1:0.5) to afford 23. Yield, 0.19 g (36%). ¹H-NMR (500MHz, CDCl₃/MeOH-d₄) δ 8.27 (s, 1H), 7.52 (d, J=2.2 Hz, 1H), 7.36 (d,J=8.5 Hz, 1H), 7.26 (dd, J=8.4, 2.2 Hz, 1H), 4.68 (d, J=6.5 Hz, 2H),3.77 (t, J=6.5 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃/MeOH-d₄) δ 154.6, 153.1,150.9, 145.0, 136.3, 135.7, 133.9, 130.3, 128.4, 128.2, 119.8, 45.0,28.5.

General Procedure for the Synthesis of 24a-m

A mixture of 23 (10 mg, 0.024 mmol) and amine (1.19 mmol, 50 equiv.) inDMF (1 mL) under nitrogen protection was stirred at room temperature for16-24 hrs. Following solvent removal, the crude material was purified bypreparative TLC (CH₂Cl₂:CH₃OH—NH₃ (7N), 20:1 or 15:1) to afford desiredproduct 24a-m.

8-((2,4-Dichlorophenyl)thio)-9-(2-(neopentylamino)ethyl)-9H-purin-6-amine(24a; HJP-V-81)

Yield, 10.1 mg (82%). ¹H-NMR (600 MHz, CDCl₃) δ 8.27 (s, 1H), 7.39 (s,1H), 7.09 (s, 2H), 5.71 (bs, 2H), 4.27-4.29 (m, 2H), 2.93 (t, J=5.8 Hz,2H), 2.26 (s, 2H), 0.77 (s, 9H); ¹³C NMR (150 MHz, CDCl₃) δ 154.7,153.3, 151.6, 144.5, 134.9, 134.5, 132.3, 130.3, 130.1, 128.1, 120.4,61.9, 49.8, 44.0, 31.6, 27.7; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₈H₂₃Cl₂N₆S, 425.1082; found 425.1081.

1-((2-(6-Amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)amino)propan-2-ol(24b; HJP-V-82)

Yield, 8.2 mg (69%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.25 (s, 1H),7.53 (s, 1H), 7.35 (d, J=8.3 Hz, 1H), 7.28 (d, J=8.4 Hz, 1H), 4.37 (t,J=5.6 Hz, 2H), 3.73-3.78 (m, 1H), 2.97-3.09 (m, 2H), 2.70-2.73 (m, 1H),2.45-2.51 (m, 1H), 1.13 (d, J=5.9 Hz, 3H); ¹³C NMR (150 MHz,CDCl₃/MeOH-d₄) δ 154.4, 152.8, 151.1, 145.3, 136.5, 135.8, 134.1, 130.4,128.2, 128.1, 119.6, 65.6, 56.5, 48.3, 43.8, 20.4; HRMS (ESI) m/z [M+H]⁺calcd. for C₁₆H₁₉Cl₂N₆OS, 413.0718; found 413.0720.

1-(2-(6-Amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)piperidin-3-ol(24c; HJP-V-83)

Yield, 8.7 mg (69%). ¹H-NMR (600 MHz, CDCl₃) δ 8.26 (s, 1H), 7.39 (d,J=2.2 Hz, 1H), 7.17 (d, J=8.5 Hz, 1H), 7.12 (dd, J=8.5, 2.2 Hz, 1H),5.84 (br s, 2H), 4.23-4.34 (m, 2H), 3.71-3.74 (m, 1H), 2.67 (t, J=4.3Hz, 2H), 2.45-2.55 (m, 3H), 2.25-2.31 (m, 1H), 1.67-1.70 (m, 1H),1.39-1.48 (m, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 154.6, 153.3, 151.5,144.5, 135.3, 134.9, 132.9, 130.1, 129.7, 128.1, 120.2, 65.8, 60.5,57.3, 54.0, 41.6, 31.3, 21.3; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₈H₂₁Cl₂N₆OS, 439.0875; found 439.0867.

2-((2-(6-Amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)amino)-2-methylpropan-1-ol(24d; HJP-V-84)

Yield, 7.3 mg (72%). ¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.25 (s, 1H),7.53 (d, J=2.2 Hz, 1H), 7.37 (d, J=8.5 Hz, 1H), 7.27 (dd, J=8.4, 2.2 Hz,1H), 4.38 (t, J=5.9 Hz, 2H), 3.32 (s, 2H), 2.96 (t, J=5.8 Hz, 2H), 1.02(s, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.6, 154.5, 152.7, 150.9, 145.5,136.7, 135.8, 134.3, 130.4, 128.2, 119.6, 67.8, 54.8, 44.4, 41.0, 23.1;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₂₁Cl₂N₆OS, 427.0875; found427.0884.

1-((2-(6-Amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)amino)-2-methylpropan-2-ol(24e; HJP-V-85)

Yield, 6.1 mg (60%). ¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.24 (s, 1H),7.53 (d, J=1.7 Hz, 1H), 7.35 (d, J=8.4 Hz, 1H), 7.28 (dd, J=8.4, 1.8 Hz,1H), 4.39 (t, J=5.4 Hz, 2H), 3.09 (t, J=5.3 Hz, 2H), 2.61 (s, 2H), 1.17(s, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.5, 152.7, 151.1, 145.3, 136.6,135.9, 134.2, 130.4, 128.3, 128.1, 119.6, 69.4, 59.9, 49.9, 43.8, 26.9;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₂₁Cl₂N₆OS, 427.0875; found427.0881.

2-((2-(6-Amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)amino)propan-1-ol(24f; HJP-V-86)

Yield, 6.5 mg (66%). ¹H NMR (600 MHz, CDCl₃) δ 8.25 (s, 1H), 7.41 (d,J=1.9 Hz, 1H), 7.11-7.17 (m, 2H), 5.78 (bs, 2H), 4.27-4.38 (m, 2H), 3.51(dd, J=11.0 and 3.7 Hz, 1H), 3.21 (dd, J=11.0 and 7.4 Hz, 1H), 3.09-3.14(m, 1H), 2.90-2.95 (m, 1H), 2.74-2.77 (m, 1H), 0.96 (d, J=6.5 Hz, 3H);¹³C NMR (150 MHz, CDCl₃) δ 154.7, 153.3, 151.6, 144.4, 135.3, 134.9,132.8, 130.2, 129.5, 128.2, 120.2, 65.5, 55.1, 46.2, 44.4, 16.9; HRMS(ESI) m/z [M+H]⁺ calcd. for C₁₆H1₉Cl₂N₆OS, 413.0718; found 413.0707.

8-((2,4-dichlorophenyl)thio)-9-(2-((2,2-difluoroethyl)amino)ethyl)-9H-purin-6-amine(24g; HJP-V-88)

Yield, 4.5 mg (57%). ¹H NMR (600 MHz, MeOD) δ 8.32 (s, 1H), 7.69 (d,J=2.3 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.41 (dd, J=8.5 and 2.3 Hz, 1H),6.29 (tt, J=53.8 and 2.8 Hz, 1H), 4.69 (t, J=5.9, 2H), 3.61-3.68 (m,4H); ¹³C NMR (150 MHz, MeOD) δ 153.8, 152.3, 149.9, 148.2, 137.9, 137.3,136.2, 131.5, 129.7, 129.1, 120.7, 114.1 (t, J=239.1 Hz), 49.7 (t,J=24.3 Hz), 48.3, 41.9; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₅H₁₅Cl₂F₂N₆OS, 418.0330; found 418.0331.

8-((2,4-Dichlorophenyl)thio)-9-(2-((2,2,2-trifluoroethyl)amino)ethyl)-9H-purin-6-amine(24h; HJP-V-89)

Yield, 6.5 mg (64%). ¹H NMR (600 MHz, CDCl₃) δ 8.27 (s, 1H), 7.40 (d,J=1.9 Hz, 1H), 7.12-7.16 (m, 2H), 5.87 (br s, 2H), 4.28 (t, J=6.1, 2H),3.05-3.11 (m, 4H); ¹³C NMR (150 MHz, CDCl₃) δ 154.1, 152.1, 151.0,145.7, 136.5, 135.8, 134.2, 130.3, 128.2, 125.3 (q, J=278.4 Hz), 119.5,51.8 (q, J=31.2 Hz), 47.9, 43.8; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₅H₁₄Cl₂F3N₆OS, 437.0330; found 437.0331.

1-(2-(6-amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)piperidin-4-ol(24i; HJP-V-90)

Yield, 4.6 mg (45%). ¹H NMR (600 MHz, MeOD) δ 8.22 (s, 1H), 7.65 (d,J=2.0 Hz, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.38 (dd, J=8.5 and 2.1 Hz, 1H),4.41-4.63 (m, 3H), 3.71-3.84 (m, 1H), 3.15-3.25 (m, 4H), 1.93-1.98 (m,2H), 1.61-1.69 (m, 2H); HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₂₁Cl₂N₆OS,439.0875; found 439.0885.

8-((2,4-Dichlorophenyl)thio)-9-(2-morpholinoethyl)-9H-purin-6-amine(24j; HJP-V-91)

Yield, 5.6 mg (55%). ¹H NMR (600 MHz, CDCl₃) δ 8.27 (s, 1H), 7.38 (d,J=2.1 Hz, 1H), 7.14 (d, J=8.5 Hz, 1H), 7.10 (dd, J=8.5 and 2.1 Hz, 1H),5.77 (br s, 2H), 4.30 (t, J=6.1, 2H), 3.56-3.59 (m, 4H), 2.66 (t, J=6.1,2H), 2.43-2.45 (m, 4H); ¹³C NMR (150 MHz, CDCl₃) δ 154.5, 153.1, 151.4,144.7, 134.8, 134.5, 132.2, 130.5, 130.1, 128.0, 120.4, 66.8, 57.6,53.8, 41.1; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₈Cl₂N₆OS, 425.0718;found 425.0716.

8-((2,4-Dichlorophenyl)thio)-9-(2-(isobutylamino)ethyl)-9H-purin-6-amine(24k; HJP-V-92)

Yield, 7.3 mg (81%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.21 (s, 1H),7.43 (d, J=2.0 Hz, 1H), 7.22 (d, J=8.0 Hz, 1H), 7.16 (dd, J=8.5, 2.0 Hz,1H), 4.31 (t, J=5.9 Hz, 2H), 2.94 (t, J=5.7, 2H), 2.34-2.37 (m, 2H),1.62-1.66 (m, 1H), 0.80 (d, J=6.5, 6H); ¹³C NMR (150 MHz, CDCl₃/MeOH-d₄)δ 154.5, 152.9, 151.2, 145.2, 136.1, 135.4, 133.7, 130.3, 128.9, 128.2,119.8, 57.3, 48.6, 43.7, 28.0, 20.4; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₂₁Cl₂N₆S, 411.0925; found 411.0917.

8-((2,4-Dichlorophenyl)thio)-9-(2-(methyl(prop-2-yn-1-yl)amino)ethyl)-9H-purin-6-amine(24l; HJP-V-100)

Yield, 6.4 mg (72%). ¹H NMR (600 MHz, CDCl₃) δ 8.25 (s, 1H), 7.40 (d,J=2.0 Hz, 1H), 7.22 (d, J=8.5 Hz, 1H), 7.14 (dd, J=8.5, 2.0 Hz, 1H),6.29 (br s, 2H), 4.31 (t, J=6.2, 2H), 3.29 (s, 2H), 2.82 (d, J=6.1 Hz,2H), 2.28 (s, 3H), 2.11 (s, 1H); ¹³C NMR (150 MHz, CDCl₃) δ 153.1,151.2, 150.2, 146.4, 135.6, 135.1, 133.3, 130.2, 129.4, 128.1, 120.0,77.6, 73.8, 54.1, 45.8, 41.9, 41.8; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₁₇Cl₂N₆S, 407.0925; found 407.0917.

1(R)-1-((2-(6-Amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)amino)propan-2-ol(24m; HJP-V-104)

Yield, 4.2 mg (53%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.22 (s, 1H),7.49 (d, J=2.2 Hz, 1H), 7.31 (d, J=8.5 Hz, 1H), 7.24 (dd, J=8.5, 2.2 Hz,1H), 4.36-4.40 (m, 2H), 3.78-3.81 (m, 1H), 2.98-3.13 (m, 2H), 2.73-2.76(m, 1H), 2.46-2.50 (m, 1H), 1.12 (d, J=6.3 Hz, 3H); ¹³C NMR (150 MHz,CDCl₃/MeOH-d₄) δ 154.6, 152.9, 151.1, 145.1, 136.5, 135.8, 134.1, 130.4,128.3, 128.2, 119.7, 65.2, 56.3, 48.2, 43.6, 20.4; HRMS (ESI) m/z [M+H]⁺calcd. for C₁₇H1₉Cl₂N₆OS, 413.0718; found 413.0729.

1(S)-1-((2-(6-Amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)amino)propan-2-ol(24n; HJP-V-105)

Yield, 3.8 mg (48%). ¹H NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.22 (s, 1H),7.49 (d, J=2.2 Hz, 1H), 7.31 (d, J=8.5 Hz, 1H), 7.24 (dd, J=8.5, 2.2 Hz,1H), 4.36-4.40 (m, 2H), 3.78-3.81 (m, 1H), 2.98-3.13 (m, 2H), 2.73-2.76(m, 1H), 2.46-2.50 (m, 1H), 1.12 (d, J=6.3 Hz, 3H); ¹³C NMR (150 MHz,CDCl₃/MeOH-d₄) δ 154.6, 152.9, 151.1, 145.1, 136.5, 135.8, 134.1, 130.4,128.3, 128.2, 119.7, 65.2, 56.3, 48.2, 43.6, 20.4; HRMS (ESI) m/z [M+H]⁺calcd. for C₁₇H₁₉Cl₂N₆OS, 413.0718; found 413.0729.

2-((2-(6-Amino-8-((2,4-dichlorophenyl)thio)-9H-purin-9-yl)ethyl)(prop-2-yn-1-yl)amino)ethanol(240; HJP-V-110)

Yield, 8.3 mg (79%). ¹H NMR (600 MHz, CD₃CN) δ 8.32 (s, 1H), 7.67 (d,J=2.1 Hz, 1H), 7.39 (d, J=8.5 Hz, 1H), 7.36 (dd, J=8.5, 2.1 Hz, 1H),4.56 (t, J=5.9, 2H), 3.94 (d, J=2.2 Hz, 2H), 3.68 (t, J=5.2, 2H), 3.43(t, J=5.9, 2H), 3.11 (t, J=5.3, 2H), 2.79 (t, J=2.2 Hz, 1H); ¹³C NMR(150 MHz, CD₃CN) δ 151.6, 150.4, 146.4, 146.1, 134.9, 134.4, 133.2,129.6, 128.8, 127.9, 119.3, 77.1, 73.8, 56.8, 55.4, 51.8, 42.1, 40.5;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₁₉Cl₂N₆OS, 437.0718; found437.0709.

6.2.8 Synthesis of Compounds of Formula 26 (Scheme 8)

9-(2-Bromoethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (25,PDP-129)

15 (1.21 mmol) was dissolved in DMF (15 mL). Cs₂CO₃ (1.45 mmol) and1,2-dibromoethane (2.42 mmol) were added and the mixture was stirredunder nitrogen at rt for 4 h. Solvent was removed under reduced pressureand the resulting residue was chromatographed (CH₂Cl₂:MeOH:AcOH,20:1:0.5) to afford 25 as a white solid in 38% yield. ¹H-NMR (400 MHz,CDCl₃) δ 8.34 (s, 1H), 7.28 (s, 2H), 7.25 (s, 1H), 6.54 (s, 2H), 4.66(t, J=6.5 Hz, 2H), 3.74 (t, J=6.5 Hz, 2H); MS (ESI): m/z 420.2 [M+H]⁺.

8-((3,5-Dichlorophenyl)thio)-9-(2-(prop-2-yn-1-ylamino)ethyl)-9H-purin-6-amine(26, PDP-131)

25 (0.09 mmol) was dissolved in DMF (5 mL). Propargylamine (0.9 mmol)was added and the mixture was stirred under nitrogen at rt for 24 h.Solvent was removed under reduced pressure and the resulting residue waschromatographed (CH₂Cl₂:NH₃/MeOH, 30:1) to afford 26 as a white solid in80% yield. ¹H-NMR (400 MHz, CDCl₃) δ 8.36 (s, 1H), 7.28 (s, 3H), 5.84(br s, 2H), 4.38 (t, J=6.1 Hz, 2H), 3.40 (d, J=2.2 Hz, 2H), 3.10 (t,J=6.1 Hz, 2H), 2.17-2.19 (m, 2H); ¹³C-NMR (CDCl₃) δ 154.8, 153.5, 151.6,143.8, 135.8, 135.3, 128.2, 127.8, 120.4, 81.5, 71.8, 47.1, 43.6, 37.7;HRMS (ESI): m/z [M+H]⁺ calcd. for C₁₆H₁₅N₆SCl₂, 393.0456; found393.0459.

6.2.9 Synthesis of Compounds of Formula 27a-d and 28a-d (Scheme 9)

General Procedure for the Synthesis of 27a-d and 28a-d

A mixture of 8-arylsulfanyl adenine (100 mmol), Cs₂CO₃ (100 mmol), and3-(tert-butoxycarbonyl-isopropyl-amino)-propyl tosylate (200 mmol) inDMF (1.3 mL) under nitrogen protection was heated at 80° C. for 30 min.Following solvent removal, the crude material was purified bypreparatory TLC with CH₂Cl₂:MeOH:AcOH at 20:1:0.1 to afford the Bocprotected N-9 and N-3 alkylated compounds. They were separately treatedwith TFA (1 ml) at 0° C. for 1.5 h to provide with corresponding9-alkyl-8-arylsulfanyladenine derivatives and3-alkyl-8-arylsulfanyladenine derivatives.

8-((2,4-Dichlorophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(27a; WS12)

¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.23 (s, 1H), 7.55 (s, 1H), 7.38 (d,J=8.3 Hz, 1H), 7.30 (d, J=8.3 Hz, 1H), 4.34 (t, J=6.7 Hz, 2H), 2.92(septet, J=5.8 Hz, 1H), 2.69 (t, J=6.4 Hz, 2H), 2.13 (pentet, J=6.5 Hz,2H), 1.16 (d, J=5.7 Hz, 6H); HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₂₁Cl₂N₆S, 411.0925; found 411.0907.

8-((2,4-Dimethylphenyl)thio)-3-(3-(isopropylamino)propyl)-3H-purin-6-amine(28b; WS11)

¹H-NMR (500 MHz, CDCl₃) δ 7.96 (s, 1H), 7.48 (d, J=7.9 Hz, 1H), 7.11 (s,1H), 7.00 (d, J=7.8 Hz, 1H), 4.48 (t, J=6.3 Hz, 2H), 2.91 (septet, J=6.3Hz, 1H), 2.65 (t, J=6.1 Hz, 2H), 2.25-2.29 (m, 2H), 1.18 (d, J=6.3 Hz,6H); HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H2₇N₆S, 371.2018; found371.2035.

8-((3,5-Dichlorophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(27c; WS13)

¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 8.21 (s, 1H), 7.26 (t, J=1.7 Hz, 1H),7.24 (d, J=1.9 Hz, 2H), 4.23 (t, J=6.9 Hz, 2H), 2.63 (septet, J=6.2 Hz,1H), 2.46 (t, J=6.4 Hz, 2H), 1.89 (pentet, J=6.9 Hz, 2H), 0.97 (d, J=6.3Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.7, 153.1, 151.3, 144.1, 135.9,133.7, 128.8, 128.7, 119.7, 48.6, 43.4, 41.6, 29.8, 22.4; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₇H₂₁Cl₂N₆S, 411.0925; found 411.0917.

8-((3,5-Dichlorophenyl)thio)-3-(3-(isopropylamino)propyl)-3H-purin-6-amine(28c; WS13-N3)

¹H-NMR (600 MHz, CDCl₃/MeOH-d₄) δ 7.98 (s, 1H), 7.33 (d, J=1.8 Hz, 2H),7.06 (t, J=1.8 Hz, 1H), 4.40 (t, J=6.7 Hz, 2H), 2.66 (septet, J=6.2 Hz,1H), 2.51 (t, J=6.4 Hz, 2H), 2.06 (pentet, J=6.5 Hz, 2H), 0.96 (d, J=6.2Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 157.9, 153.4, 151.0, 142.8, 137.8,134.9, 127.9, 126.8, 122.6, 48.9, 48.1, 43.0, 29.5, 22.9; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₇H₂₁Cl₂N₆S, 411.0925; found 411.0928.

8-((3,5-Bis(trifluoromethyl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(27d; WS14)

¹H-NMR (500 MHz, CDCl₃) δ 8.33 (s, 1H), 7.26-7.28 (m, 3H), 5.77 (br s,2H), 4.33 (t, J=7.0 Hz, 2H), 2.71-2.76 (m, 1H), 2.57 (t, J=6.9 Hz, 2H),1.96-1.99 (m, 2H), 1.05 (d, J=6.4 Hz, 6H); HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₉H₂₁F6N₆S, 479.1453; found 479.1444.

6.2.10 Synthesis of Compounds of Formula 30a-n (Scheme 10)

General Procedure for the Synthesis of 30a-n

A mixture of 8-arylsulfanyl adenine (29a-n; 100 mmol), Cs₂CO₃ (100mmol), and pent-4-ynyl-4-methylbenzenesulfonate (120 mmol) in DMF (1.3mL) under nitrogen protection was heated at 80° C. for 30 min. Followingsolvent removal, the crude material was purified by preparatory TLC(CHCl₃:MeOH:NH₄OH, 10:1:0.5 or CHCl₃:MeOH:AcOH, 10:1:0.5) to provide thecorresponding 3-alkyl-8-arylsulfanyladenine derivatives 30a-n.

8-(2-Chlorophenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30a)

Yield, 10%. ¹H NMR (400 MHz, CDCl₃) δ 8.02 (s, 1H), 7.48-7.51 (m, 1H),7.40-7.42 (m, 1H), 7.14-7.17 (m, 2H), 4.49-4.51 (m, 2H), 2.17-2.22 (m,4H), 2.05-2.06 (m, 1H); HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₅N₅ClS,344.0737; found 344.0720.

8-(2-Methoxyphenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30b)

Yield, 25%. ¹H NMR (400 MHz, CDCl₃) δ 7.97 (s, 1H), 7.45 (d, J=7.7 Hz,1H), 7.22 (d, J=7.8 Hz, 1H), 6.86-6.90 (m, 2H), 4.47 (t, J=6.6 Hz, 2H),3.87 (s, 3H), 2.21-2.26 (m, 4H), 2.05-2.07 (m, 1H); ¹³C NMR (100 MHz,CDCl₃) δ 157.7, 152.6, 141.8, 132.5, 128.3, 121.1, 111.1, 81.6, 70.5,55.9, 49.0, 27.0, 15.3; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H1₈N₅OS,340.1232; found 340.1218.

(2-(6-Amino-3-(pent-4-ynyl)-3H-purin-8-ylthio)phenyl)methanol (30c)

Yield, 21%.

¹H NMR (400 MHz, CDCl₃) δ 7.96 (s, 1H), 7.74 (d, J=7.6 Hz, 1H), 7.60 (d,J=7.5 Hz, 1H), 7.44 (t, J=7.4 Hz, 1H), 7.29 (t, J=7.5 Hz, 1H), 4.92 (s,2H), 4.40 (t, J=6.4 Hz, 2H), 3.47 (br s, 1H), 2.11-2.20 (m, 4H),2.03-2.06 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 146.2, 142.6, 137.3,131.5, 130.9, 129.1, 82.1, 71.2, 64.9, 49.6, 27.1, 15.6; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₇H₁₈N₅OS, 340.1232; found 340.1242.

3-(Pent-4-ynyl)-8-(2-(trifluoromethoxy)phenylthio)-3H-purin-6-amine(30d)

Yield, 19%. ¹H NMR (400 MHz, CDCl₃) δ 8.01 (s, 1H), 7.50 (d, J=7.9 Hz,1H), 7.21-7.27 (m, 2H), 7.13 (t, J=7.1 Hz, 1H), 4.48 (t, J=6.1 Hz, 2H),2.17-2.20 (m, 4H), 2.04-2.05 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 158.3,153.1, 142.2, 132.1, 128.1, 127.9, 126.9, 122.5, 120.7, 81.8, 70.5,49.0, 26.9, 15.2; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₅N₅F₃OS,394.0949; found 394.0946.

8-(2,4-Dichlorophenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30e)

Yield, 22%. ¹H NMR (400 MHz, CDCl₃/MeOH-d₄) δ 8.10 (s, 1H), 7.50 (s,1H), 7.42 (d, J=8.4 Hz, 1H), 7.23-7.25 (m, 1H), 4.46 (t, J=6.5 Hz, 2H),2.24-2.27 (m, 2H), 2.14-2.19 (m, 3H); ¹³C NMR (100 MHz, CDCl₃/MeOH-d₄) δ154.8, 152.9, 150.0, 142.8, 133.9, 132.3, 130.6, 129.3, 128.8, 127.2,120.4, 81.3, 70.0, 49.8, 26.7, 14.7; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₆H₁₄N₅Cl₂S, 378.0347; found 378.0335.

8-(2,4-Dimethylphenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30f)

Yield, 27%. ¹H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.48 (d, J=7.7 Hz,1H), 7.08 (s, 1H), 6.97 (d, J=7.7 Hz, 1H), 4.36 (t, J=6.1 Hz, 2H), 2.51(s, 3H), 2.39 (s, 3H), 2.15-2.19 (m, 4H), 2.03-2.05 (m, 1H); ¹³C NMR(100 MHz, CDCl₃) δ 162.6, 151.9, 151.1, 141.5, 141.1, 138.6, 134.5,131.3, 127.3, 127.2, 121.7, 81.8, 70.5, 48.8, 26.9, 21.1, 20.8, 15.2;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₂₀N₅S, 338.1439; found 338.1427.

8-(2,4-Dimethoxyphenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30g)

Yield, 7%. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (s, 1H), 7.53 (d, J=8.1 Hz,1H), 6.56-6.59 (m, 2H), 4.43 (t, J=6.8 Hz, 2H), 3.86 (s, 3H), 3.82 (s,3H), 2.12-2.26 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 162.2, 160.6, 151.8,141.6, 137.3, 135.2, 108.8, 105.3, 99.1, 81.5, 70.1, 55.6, 55.1, 26.7,14.8; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₂₀N₅O₂S, 370.1338; found370.1350.

8-(2,5-Dichlorophenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30h)

Yield, 21%. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (s, 1H), 7.43 (s, 1H),7.26-7.28 (m, 1H), 7.06-7.08 (m, 1H), 4.50-4.52 (m, 2H), 2.21-2.24 (m,4H), 2.05-2.06 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 153.6, 142.6, 132.7,130.3, 129.7, 127.3, 81.7, 70.6, 49.2, 26.9, 15.2; HRMS (ESI) m/z [M+H]⁺calcd. for C₁₆H₁₄N₅Cl₂S, 378.0347; found 378.0362.

8-(2,5-Dimethylphenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30i)

Yield, 20%. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (s, 1H), 7.41 (s, 1H), 7.13(d, J=7.7 Hz, 1H), 7.04 (d, J=7.7 Hz, 1H), 4.45 (t, J=6.3 Hz, 2H), 2.43(s, 3H), 2.37 (s, 3H), 2.17-2.27 (m, 3H), 2.04 (m, 2H); ¹³C NMR (100MHz, CDCl₃) δ 152.0, 151.2, 141.7, 137.7, 135.9, 134.5, 130.9, 130.2,129.2, 81.8, 70.5, 48.9, 27.0, 20.8, 20.4, 15.3; HRMS (ESI) m/z [M+H]⁺calcd. for C₁₈H₂₀N₅S, 338.1439; found 338.1435.

8-(2-Chloro-5-(trifluoromethyl)phenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine(30j)

Yield, 18%. H NMR (400 MHz, CDCl₃/MeOH-d⁴) δ 8.08 (s, 1H), 7.84 (s, 1H),7.57 (d, J=8.3 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H), 4.48 (t, J=6.5 Hz, 2H),2.16-2.26 (m, 4H), 2.09-2.10 (m, 1H); ¹³C NMR (100 MHz, CDCl₃/MeOH-d4) δ157.4, 153.2, 150.4, 142.8, 138.2, 134.5, 130.1, 129.4, 128.9, 124.8,121.9, 81.5, 70.4, 28.3, 26.7, 15.0; MS (ESI): m/z 411.8 [M+H]⁺.

8-(3,5-Dichlorophenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30k)

Yield, 14%. ¹H NMR (400 MHz, CDCl₃) δ 8.03 (s, 1H), 7.46 (s, 2H), 7.30(s, 1H), 4.50-4.52 (m, 2H), 2.20-2.24 (m, 3H), 2.04-2.07 (m, 2H); ¹³CNMR (100 MHz, CDCl₃/MeOH-d₄) δ 157.6, 152.6, 150.8, 142.9, 136.3, 134.8,128.7, 127.1, 120.4, 81.3, 70.1, 43.8, 26.7, 14.7; HRMS (ESI) m/z [M+H]⁺calcd. for C₁₄H₁₅N₅Cl₂S, 378.0347; found 378.0340.

8-(3,5-Dimethylphenylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30l)

Yield, 16%. ¹H NMR (400 MHz, CDCl₃/MeOH-d₄) δ 8.01 (s, 1H), 7.23 (s,2H), 7.00 (s, 1H), 4.45 (t, J=7.5 Hz, 2H), 2.26 (s, 6H), 2.21-2.25 (m,4H), 1.94-1.99 (m, 1H); HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₂₀N₅S,338.1439; found 338.1426.

3-(Pent-4-ynyl)-8-(2,4,5-trichlorophenylthio)-3H-purin-6-amine (30m)

Yield, 23%. ¹H NMR (400 MHz, CDCl₃/MeOH-d₄) δ 8.04 (s, 1H), 7.73 (s,1H), 7.55 (s, 1H), 4.50 (t, J=6.4 Hz, 2H), 2.08-2.29 (m, 4H), 2.06-2.07(m, 1H); ¹³C NMR (100 MHz, CDCl₃/MeOH-d₄) δ 159.6, 154.7, 152.4, 144.3,135.3, 134.9, 134.7, 133.5, 132.9, 132.4, 128.8, 83.4, 72.2, 48.6, 28.6,16.8; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₃N₅Cl₃S, 411.9957; found411.9947.

8-(Mesitylthio)-3-(pent-4-ynyl)-3H-purin-6-amine (30n)

Yield, 15% ¹H NMR (400 MHz, CDCl₃/MeOH-d₄) δ 7.93 (s, 1H), 6.97 (s, 2H),4.38 (t, J=6.5 Hz, 2H), 2.41 (s, 6H), 2.30 (s, 3H), 2.04-2.16 (m, 5H);¹³C NMR (100 MHz, CDCl₃/MeOH-d₄) δ 157.4, 150.4, 143.0, 142.2, 139.2,128.9, 124.8, 81.5, 70.1, 26.7, 21.6, 21.3, 20.5, 14.8; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₉H₂₂N₅S, 352.1596; found 352.1594.

6.2.11 Synthesis of Compounds of Formula 32-34 (Scheme 11)

8-((3-chloro-5-iodophenyl)thio)-9H-purin-6-amine (31)

8-Mercaptoadenine (3.6 mmol), neocuproine hydrate (0.36 mmol), CuI (0.36mmol), NaO-t-Bu (7.2 mmol), 1-chloro-3,5-diiodobenzene (10.8 mmol), andanhydrous DMF (24 mL) were taken in a round bottom flask flushed withnitrogen. The flask was sealed with Teflon tape, heated at 110° C., andmagnetically stirred for 24 h under nitrogen. Solvent was removed underreduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5). Obtained as a light yellow solid in 67%yield. MS (ESI): m/z 403.7 [M+H]⁺. Compound 15 was made in a similarmanner.

General Procedure for the Synthesis of 32-34

To a suspension of coupled product (15 or 31, 1.0 mmol) inCH₂Cl₂:toluene (0.5:2.5 mL) were added PPh₃ (4.0 mmol) and alcohol (2.0mmol) under nitrogen protection. After stirring for 10 min. DBAD (6mmol) was added and reaction mixture was stirred at rt for 2-5 h.Following solvent removal, the crude material was purified bypreparative TLC (CH₂Cl₂:CH₃OH:AcOH, 20:1:0.1 or CH₂Cl₂:NH₃—CH₃OH (7N),20:1) to afford desired compounds 32-34.

8-((3,5-dichlorophenyl)thio)-9-(hex-5-yn-3-yl)-9H-purin-6-amine (32,HJP-V-125)

Yield, 9.2 mg (37%). ¹H NMR (600 MHz, CDCl₃) δ 8.25 (s, 1H), 7.35-7.37(m, 2H), 7.33 (t, J=1.7 Hz, 1H), 4.71-4.74 (m, 1H), 3.27-3.33 (m, 1H),2.80-2.84 (m, 1H), 2.32-2.35 (m, 1H), 2.02-2.05 (m, 1H), 1.84 (t, J=2.5Hz, 1H), 0.79 (t, J=7.4 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 153.6,150.5, 147.7, 135.8, 133.6, 132.1, 131.9, 129.3, 128.9, 128.4, 79.6,71.1, 59.9, 26.2, 23.3, 10.9; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₁₅Cl₂N₅S, 392.0503; found 392.0503.

8-((3,5-dichlorophenyl)thio)-9-(2-(pyridin-3-yl)ethyl)-9H-purin-6-amine(33, HJP-V-140)

Yield, 9.6 mg (36.9%). ¹H NMR (600 MHz, CDCl₃) δ 8.33-8.47 (m, 2H), 8.20(s, 1H), 7.69 (d, J=7.4 Hz, 1H), 7.45-7.55 (m, 1H), 7.39 (t, J=1.7 Hz,1H), 7.35-7.37 (m, 2H), 4.55 (t, J=7.2 Hz, 2H), 3.27 (t, J=7.0, Hz, 2H);¹³C NMR (150 MHz, CDCl₃) δ 150.9, 150.3, 148.8, 146.8, 145.6, 144.9,139.6, 136.1, 130.9, 130.6, 130.4, 130.0, 119.3, 44.8, 32.8′; HRMS (ESI)m/z [M+H]⁺ calcd. for C₁₈H₁₅Cl₂N₆S, 417.0456; found 417.0446.

8-((3-chloro-5-iodophenyl)thio)-9-(2-(pyridin-3-yl)ethyl)-9H-purin-6-amine(34, HJP-V-147)

Yield, 8.1 mg (32%). ¹H NMR (600 MHz, CD₃CN) δ 8.57 (d, J=5.5 Hz, 1H),8.50 (s, 1H), 8.14 (d, J=8.0 Hz, 1H), 7.80 (t, J=1.6 Hz, 1H), 7.70-7.78(m, 1H), 7.73 (t, J=1.5 Hz, 1H), 7.47 (t, J=1.7 Hz, 1H), 4.58 (t, J=7.4Hz, 2H), 3.34 (t, J=6.4, Hz, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 150.5,150.1, 147.1, 146.1, 144.2, 141.8, 140.4, 137.2, 137.1, 136.8, 134.9,132.8, 129.8, 126.5, 119.3, 93.8, 44.1, 31.5; HRMS (ESI) m/z [M+H]⁺calcd. for C₁₈H₁₅IClN₆S, 508.9812; found 508.9826.

6.2.12 Synthesis of Compounds of Formula 36-48 (Scheme 12)

9-(3-bromopropyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (35)

Synthesis of compound 35 was done in a manner similar to that ofcompound 25, except the 1,2-dibromoethane was substituted by1,3-dibromopropane. Following solvent removal, the crude material waspurified by preparatory TLC (CH₂Cl₂:MeOH:AcOH, 20:1:0.1) to providedesired isomer 35. ¹H-NMR (600 MHz, CDCl₃) δ 8.36 (s, 1H), 7.26-7.31 (m,3H), 5.68 (br s, 2H), 4.31 (t, J=7.3 Hz, 2H), 3.15 (t, J=6.7 Hz, 2H),2.28-2.35 (m, 2H); MS (ESI): m z 432.1 [M+H]⁺.

General Procedure for the Synthesis of 36-40

A mixture of 35 (0.027 mmol) and amine (1.35 mmol, 50 equiv.) in DMF (1mL) under nitrogen protection was stirred at room temperature for 16-24hrs. Following solvent removal, the crude material was purified bypreparative TLC (CH₂Cl₂:CH₃OH—NH₃ (7N), 20:1 or 15:1) to afford desiredproducts 36-40.

1-((3-(6-amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)amino)propan-2-ol(36, HJP-V-130)

Yield, 6.1 mg (76%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.22 (s,1H), 7.28-7.33 (m, 3H), 4.27 (t, J=6.9 Hz, 2H), 3.77-3.83 (m, 1H),2.51-2.62 (m, 3H), 2.37-2.42 (m, 1H), 1.93-1.95 (m, 2H), 1.11 (d, J=6.2Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 154.6, 153.1, 151.2, 144.4, 135.9,133.3, 129.1, 128.9, 119.6, 65.4, 56.5, 45.7, 41.4, 29.4, 20.6; HRMS(ESI) m/z [M+H]⁺ calcd. for C1₇H₂₁Cl₂N₆OS, 427.0875; found 427.0887.

2-((3-(6-amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)(prop-2-yn-1-yl)amino)ethanol(37, HJP-V-132)

Yield, 6.2 mg (60%). ¹H NMR (600 MHz, CD₃CN) δ 8.31 (s, 1H), 7.48 (t,J=1.6 Hz, 1H), 7.49-7.51 (m, 2H), 4.33 (t, J=6.5 Hz, 2H), 3.99-4.05 (m,2H), 3.78-3.81 (m, 2H), 3.18-3.21 (m, 4H), 2.90 (s, 1H), 2.22-2.28 (m,2H); ¹³C NMR (150 MHz, CD₃CN) δ 151.3, 150.5, 146.3, 145.9, 135.1,133.6, 128.7, 128.2, 117.8, 79.1, 71.2, 55.6, 54.9, 50.4, 42.0, 40.8,23.7; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H₂₁Cl₂N₆OS, 451.0875; found451.0879.

1-(3-(6-amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)azetidin-3-ol(38, HJP-V-134)

Yield, 4.2 mg (36%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.27 (s,1H), 7.37 (t, J=1.7 Hz, 1H), 7.32-7.34 (m, 2H), 4.38-4.44 (m, 1H), 4.28(t, J=7.2 Hz, 2H), 3.7 (t, J=7.2 Hz, 2H), 3.05 (t, J=7.2 Hz, 2H),2.59-2.63 (m, 2H), 1.87-1.93 (m, 2H); ¹³C NMR (150 MHz, CDCl₃+5 dropsCD₃OD) δ 154.7, 153.2, 151.2, 144.6, 136.0, 122.6, 129.2, 129.0, 119.8,63.8, 61.4, 55.7, 41.8, 27.2; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H1₉Cl₂N₆OS, 425.0718; found 425.0718.

(S)-9-(3-((1-cyclopropylethyl)amino)propyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine(39, SO-III-127B)

9-(3-bromopropyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (12 mg,0.027 mmol) in dry DMF (1 mL) was added (S)-1-cyclopropylethanamine(58.3 μL, 0.554 mmol) and then the reaction mixture was stirred at rtfor 4 days. Solvent was removed under reduced pressure and the residuewas purified by preparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 15:1) to afford6.0 mg (51%) of SO-III-127B. ¹H NMR (600 MHz, CDCl₃): δ 8.36 (s, 1H),7.27-7.30 (m, 3H), 5.68 (br s, 2H), 4.31-4.34 (m, 2H), 2.55-2.68 (m,2H), 1.95-1.99 (m, 2H), 1.75-1.79 (m, 1H), 1.11 (d, J=6.3 Hz, 3H),0.64-0.69 (m, 1H), 0.46-0.50 (m, 1H), 0.39-0.42 (m, 1H), 0.13-0.17 (m,1H), 0.02-0.06 (m, 1H). ¹³C NMR (150 MHz, CDCl₃): δ 154.6, 153.4, 151.6,143.6, 135.8, 134.7, 128.4, 128.0, 120.3, 58.9, 43.9, 41.8, 30.4, 20.6,17.7, 4.6, 1.7. HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H2₃N₆SCl₂, 437.1082;found 437.1083.

(R)-9-(3-((1-cyclopropylethyl)amino)propyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine(40, SO-III-128B)

9-(3-bromopropyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (12 mg,0.027 mmol) in dry DMF (1 mL) was added (R)-1-cyclopropylethanamine(58.3 μL, 0.554 mmol) and then the reaction mixture was stirred at rtfor 4 days. Solvent was removed under reduced pressure and the residuewas purified by preparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 15:1) to afford6.1 mg (52%) of SO-III-128B. ¹H NMR (600 MHz, CDCl₃): δ 8.36 (s, 1H),7.27-7.30 (m, 3H), 5.68 (br s, 2H), 4.31-4.34 (m, 2H), 2.55-2.68 (m,2H), 1.95-1.99 (m, 2H), 1.75-1.79 (m, 1H), 1.11 (d, J=6.3 Hz, 3H),0.64-0.69 (m, 1H), 0.46-0.50 (m, 1H), 0.39-0.42 (m, 1H), 0.13-0.17 (m,1H), 0.02-0.06 (m, 1H). ¹³C NMR (150 MHz, CDCl₃): δ 154.6, 153.4, 151.6,143.6, 135.8, 134.7, 128.4, 128.0, 120.3, 58.9, 43.9, 41.8, 30.4, 20.6,17.7, 4.6, 1.7. HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H2₃N₆SCl₂, 437.1082;found 437.1077.

9-(3-(1H-imidazol-1-yl)propyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine(41, SO-III-103A)

To 8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (60 mg, 0.192 mmol) indry DMF (3 mL) was added Cs₂CO₃ (75 mg, 0.230 mmol) and1-(3-bromopropyl)-1H-imidazole (181 mg, 0.96 mmol) and then the reactionmixture was stirred at rt for 2 hours. Then another portion of Cs₂CO₃(20 mg) was added to the reaction mixture which was further stirred forone more hour. Solvent was removed under reduced pressure and theresidue was purified by preparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 10:1) toafford 4.9 mg (6%) of SO-III-103A. ¹H NMR (600 MHz, CDCl₃): δ 8.38 (s,1H), 7.64 (s, 1H), 7.32 (t, J=1.8 Hz, 1H), 7.24 (d, J=1.8 Hz, 2H), 7.12(m, 1H), 6.97 (m, 1H), 5.68 (br s, 2H), 4.23 (t, J=7.0 Hz, 2H), 4.02 (t,J=7.0 Hz, 2H), 2.27 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 154.7, 153.7,151.6, 143.3, 137.1, 135.9, 134.0, 129.6, 128.7, 128.2, 120.2, 118.7,44.2, 40.9, 31.1. HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₁₆N₇SCl₂,420.0565; found 420.0555.

9-(2-(cyclopropylamino)ethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine(42, SO-III-35A)

To 9-(2-bromoethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (10mg, 0.0238 mmol) in dry DMF (1 mL) was added cyclopropylamine (8.26 μL,0.119 mmol) and then the reaction mixture was stirred at rt for 24 h.Then to the reaction mixture was added more cyclopropylamine (17.3 μL,0.25 mmol) and the reaction was further stirred at rt for 48 h. Solventwas removed under reduced pressure and the residue was purified bypreparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 30:1) to afford 6.0 mg (64%) ofSO-III-35A. ¹H NMR (600 MHz, CDCl₃): δ 8.37 (s, 1H), 7.27-7.29 (m, 3H),5.6 (br s, 2H), 4.35 (t, J=6.4 Hz, 2H), 3.08 (t, J=6.4 Hz, 2H), 2.14 (m,1H), 0.39 (m, 2H), 0.23 (m, 2H). ¹³C NMR (150 MHz, CDCl₃): δ 154.7,153.5, 151.6, 144.0, 135.7, 135.1, 128.2, 127.9, 120.3, 48.4, 44.1,30.1, 6.5. HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H1₇N₆SCl₂, 395.0612;found 395.0626.

(R)-9-(2-((1-cyclopropylethyl)amino)ethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine(43, SO-III-36A)

To 9-(2-bromoethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (10mg, 0.0238 mmol) in dry DMF (1 mL) was added (R)-1-cyclopropylethanamine(12.7 μL, 0.119 mmol) and then the reaction mixture was stirred at rtfor 24 h. Then to the reaction mixture was added more(R)-1-cyclopropylethanamine (12.7 μL, 0.119 mmol) and the reaction wasfurther stirred at rt for 24 h. Solvent was removed under reducedpressure and the residue was purified by preparatory TLC(CH₂Cl₂:MeOH—NH₃ (7N), 30:1) to afford 5.7 mg (57%) of SO-III-36A. ¹HNMR (500 MHz, CDCl₃:CD₃OD): δ 8.27 (s, 1H), 7.33-7.35 (m, 3H), 4.35 (t,J=6.6 Hz, 2H), 3.03-3.07 (m, 1H), 2.95-3.0 (m, 1H), 1.87 (m, 1H), 1.09(d, J=6.3 Hz, 3H), 0.63 (m, 1H), 0.40-0.47 (m, 2H), 0.13 (m, 1H), 0.04(m, 1H). ¹³C NMR (150 MHz, CDCl₃:CD₃OD): δ 154.8, 153.2, 151.3, 144.9,136.0, 134.1, 129.1, 128.9, 119.9, 58.6, 46.1, 44.2, 20.3, 17.3, 4.6,1.7. HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₂₁N₆SCl₂, 423.0925; found423.0909.

(S)-9-(2-((1-cyclopropylethyl)amino)ethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine(44, SO-III-37A)

To 9-(2-bromoethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (10mg, 0.0238 mmol) in dry DMF (1 mL) was added (S)-1-cyclopropylethanamine(12.7 μL, 0.119 mmol) and then the reaction mixture was stirred at rtfor 24 h. Then to the reaction mixture was added more(S)-1-cyclopropylethanamine (12.7 μL, 0.119 mmol) and the reaction wasfurther stirred at rt for 24 h. Solvent was removed under reducedpressure and the residue was purified by preparatory TLC(CH₂Cl₂:MeOH—NH₃ (7N), 30:1) to afford 6.4 mg (64%) of SO-III-37A. ¹HNMR (500 MHz, CDCl₃:CD₃OD): δ 8.27 (s, 1H), 7.33-7.35 (m, 3H), 4.35 (t,J=6.6 Hz, 2H), 3.03-3.07 (m, 1H), 2.95-3.0 (m, 1H), 1.87 (m, 1H), 1.09(d, J=6.3 Hz, 3H), 0.63 (m, 1H), 0.40-0.47 (m, 2H), 0.13 (m, 1H), 0.04(m, 1H). ¹³C NMR (150 MHz, CDCl₃:CD₃OD): δ 154.8, 153.2, 151.3, 144.9,136.0, 134.1, 129.1, 128.9, 119.9, 58.6, 46.1, 44.2, 20.3, 17.3, 4.6,1.7. HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₂₁N₆SCl₂, 423.0925; found423.0909.

8-((3,5-dichlorophenyl)thio)-9-(2-(4-methylpiperazin-1-yl)ethyl)-9H-purin-6-amine(45, SO-III-39A)

To 9-(2-bromoethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (10mg, 0.0238 mmol) in dry DMF (1 mL) was added 1-methylpiperazine (51 μL,0.46 mmol) and then the reaction mixture was stirred at rt for 48 h.Solvent was removed under reduced pressure and the residue was purifiedby preparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 15:1) to afford 8 mg (77%) ofSO-III-39A. ¹H NMR (600 MHz, CDCl₃): δ 8.36 (s, 1H), 7.27 (m, 1H), 7.24(m, 2H), 5.69 (br s, 2H), 4.35 (t, J=6.2 Hz, 2H), 2.71 (t, J=6.2 Hz,2H), 2.48-2.58 (m, 4H), 2.32-2.42 (m, 4H), 2.26 (s, 3H). ¹³C NMR (150MHz, CDCl₃): δ 154.7, 153.4, 151.4, 144.0, 135.8, 135.7, 128.0, 127.6,120.5, 56.9, 54.9, 53.2, 45.9, 41.4. HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₈H2₂N₇SCl₂, 438.1034; found 438.1024.

9-(2-((2-cyclopropylpropan-2-yl)amino)ethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine(46, SO-III-40A)

To 9-(2-bromoethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (10mg, 0.023 mmol) in dry DMF (1 mL) was added 2-Cyclopropyl-2-propylaminep-toluenesulfonate salt (125 mg, 0.46 mmol) and Et₃N (50 μL) then thereaction mixture was stirred at rt for 10 days. Solvent was removedunder reduced pressure and the residue was purified by preparatory TLC(CH₂Cl₂:MeOH—NH₃ (7N), 10:1) to afford 3.8 mg (38%) of SO-III-40A. ¹HNMR (600 MHz, CDCl₃:CD₃OD): δ 8.26 (s, 1H), 7.35-7.37 (m, 3H), 4.47 (m,2H), 2.99 (m, 2H), 0.91 (s br, 6H), 0.88 (m, 1H), 0.39 (m, 2H), 0.24 (m,2H). HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H2₃N₆SCl₂, 437.1082; found437.1090.

8-((3,5-dichlorophenyl)thio)-9-(2-((2-methoxypropyl)amino)ethyl)-9H-purin-6-amine(47, SO-III-75A)

To 9-(2-bromoethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (10mg, 0.023 mmol) in dry DMF (2 mL) was added 2-methoxy-1-propanaminehydrochloride (24.5 mg, 0.195 mmol), Et₃N (50 μL) and the reactionmixture was stirred at rt for 3 days. Solvent was removed under reducedpressure and the residue was purified by preparatory TLC(CH₂Cl₂:MeOH—NH₃ (7N), 20:1) to afford 9.0 mg (92%) of SO-III-75A. ¹HNMR (600 MHz, CDCl₃): δ 8.36 (s, 1H), 7.25-7.28 (m, 3H), 5.93 (br s,2H), 4.35 (t, J=6.4 Hz, 2H), 3.34-3.37 (m, 1H), 3.29 (s, 3H), 2.95-3.03(m, 2H), 2.55-2.58 (m, 2H), 1.07 (d, J=6.2 Hz, 3H). ¹³C NMR (150 MHz,CDCl₃): δ 154.8, 153.4, 151.5, 144.0, 135.7, 135.3, 128.1, 127.8, 120.4,75.9, 56.2, 55.1, 48.7, 43.9, 16.9. HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₂₁N₆OSCl₂, 427.0875; found 427.0860.

8-((3,5-dichlorophenyl)thio)-9-(2-(isopropylamino)ethyl)-9H-purin-6-amine(48, SO-III-116A)

To 9-(2-bromoethyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (20mg, 0.047 mmol) in dry DMF (2 mL) was added isopropylamine (121 μL, 1.41mmol) and the reaction mixture was stirred at rt for 72 hours. Solventwas removed under reduced pressure and the residue was purified bypreparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 20:1) to afford 12.6 mg (68%) ofSO-III-116A. ¹H NMR (600 MHz, CDCl₃): δ 8.36 (s, 1H), 7.26-7.28 (m, 3H),5.82 (br s, 2H), 4.34 (t, J=6.5 Hz, 2H), 2.97 (t, J=6.5 Hz, 2H),2.74-2.76 (m, 1H), 0.96 (d, J=6.2 Hz, 6H). ¹³C NMR (150 MHz, CDCl₃): δ154.8, 153.5, 151.5, 144.0, 135.7, 135.3, 128.1, 127.8, 120.4, 48.5,46.1, 44.5, 22.8. HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₆H₁₉N₆SCl₂,397.0769; found 397.0765.

6.2.13 Synthesis of Compounds of Formula 51 (Scheme 13a)

8-((2,6-dichloropyridin-4-yl)thio)-9H-purin-6-amine (50)

8-Mercaptoadenine (3.6 mmol), neocuproine hydrate (0.36 mmol), CuI (0.36mmol), NaO-t-Bu (7.2 mmol), 2,6-dichloro-4-iodopyridine (10.8 mmol), andanhydrous DMF (24 mL) were taken in a round bottom flask flushed withnitrogen. The flask was sealed with Teflon tape, heated at 110° C., andmagnetically stirred for 24 h under nitrogen. Solvent was removed underreduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5). Obtained as a light yellow solid in 50%yield. MS (ESI): m/z 312.8 [M+H]⁺.

8-((2,6-dichloropyridin-4-yl)thio)-9-(pent-4-yn-1-yl)-9H-purin-6-amine(51, HJP-VI-66)

8-Arylsulfanyl adenine (1.21 mmol) was dissolved in DMF (15 mL) andCs₂CO₃ (1.45 mmol) and 5-chloropent-1-yne (2.42 mmol) were added and themixture was stirred under nitrogen at for 3 h. Solvent was removed underreduced pressure and the resulting residue was purified by Preparativechromatography (CH₂Cl₂:MeOH:AcOH, 20:1:0.5) to afford desired compoundHJP-VI-66. Obtained as a solid in 22% yield. ¹H NMR (600 MHz, CDCl₃+5drops of CD₃OD): δ 8.32 (s, 1H), 7.47 (s, 1H), 7.18 (s, 1H), 4.38 (t,J=7.2 Hz, 2H), 2.26-2.29 (m, 2H), 2.03-2.05 (m, 3H), 0.13 (m, 1H), 0.04(m, 1H). ¹³C NMR (150 MHz, CDCl₃:CD₃OD): δ 159.3, 157.6, 155, 154.9,153.9, 124.4, 124.3, 85.8, 73.6, 47.3, 41.7, 32.3, 19.7; HRMS (ESI) m/z[M+H]⁺ calcd. for C₁₅H1₃N₆SCl₂, 379.0299; found 379.0312.

6.2.13 Synthesis of Compounds of Formula 53-56 (Scheme 13b)

9-(3-bromopropyl)-8-((3-chloro-5-iodophenyl)thio)-9H-purin-6-amine (52)

8-Arylsulfanyl adenine (1.21 mmol) was dissolved in DMF (15 mL) andCs₂CO₃ (1.45 mmol) and 1,3-dibromopropane (2.42 mmol) were added and themixture was stirred under nitrogen at for 2-4 h. Solvent was removedunder reduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5) to afford desired compound 52. Obtained asa solid in 25% yield. MS (ESI): m/z 523.9 [M+H]⁺.

General Procedure for the Synthesis of 53-56

A mixture of 52 (12 mg, 0.028 mmol) and amine (1.40 mmol, 50 equiv.) inDMF (1 mL) under nitrogen protection was stirred at room temperature for16-24 hrs. Following solvent removal, the crude material was purified bypreparative TLC (CH₂Cl₂:CH₃OH—NH₃ (7N), 20:1 or 15:1) to afford desiredproducts 53-56.

8-((3-chloro-5-iodophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(53, HJP-V-149)

Yield, 9.5 mg (83%). ¹H NMR (600 MHz, CDCl₃) δ 8.34 (s, 1H), 7.62 (t,J=1.4 Hz, 1H), 7.60 (t, J=1.6 Hz, 1H), 7.34 (t, J=1.7 Hz, 1H), 6.13 (brs, 2H), 4.32 (t, J=7.0 Hz, 2H), 2.67-2.73 (m, 1H), 2.55 (t, J=6.7 Hz,2H), 1.92-1.98 (m, 2H), 1.03 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃)δ 154.8, 153.4, 151.6, 143.6, 136.7, 136.6, 135.7, 134.7, 129.3, 120.2,94.4, 48.7, 43.8, 41.8, 30.3, 22.9; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₂₁IClN₆S, 503.0282; found 503.0260.

8-((3-chloro-5-iodophenyl)thio)-9-(3-(isobutylamino)propyl)-9H-purin-6-amine(54, HJP-VI-4)

Yield, 10.2 mg (85%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.26 (s,1H), 7.70 (t, J=1.6 Hz, 1H), 7.68 (t, J=1.6 Hz, 1H), 7.40 (t, J=1.7 Hz,1H), 4.30 (t, J=7.0 Hz, 2H), 2.55 (t, J=6.9, Hz, 2H), 2.35 (d, J=6.9 Hz,2H), 1.95-2.01 (m, 2H), 1.72-1.79 (m, 1H), 0.92 (d, J=6.7 Hz, 6H); HRMS(ESI) m/z [M+H]⁺ calcd. for C₁₈H₂₃IClN₆S, 517.0438; found 517.0457.

8-((3-chloro-5-iodophenyl)thio)-9-(3-(neopentylamino)propyl)-9H-purin-6-amine(55, HJP-VI-5)

Yield, 10.3 mg (85%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.24 (s,1H), 7.72 (t, J=1.5 Hz, 1H), 7.69 (t, J=1.4 Hz, 1H), 7.41 (t, J=1.6 Hz,1H), 4.31 (t, J=7.0 Hz, 2H), 2.60 (t, J=6.8 Hz, 2H), 2.33 (s, 2H),1.99-2.05 (m, 2H), 0.95 (s, 9H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD)δ 158.6, 156.8, 155.1, 148.6, 141.7, 141.3, 139.8, 137.3, 134.3, 123.6,65.8, 50.8, 45.5, 35.1, 32.9, 31.6; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₉H₂₅IClN₆S, 531.0595; found 531.0587.

9-(3-(tert-butylamino)propyl)-8-((3-chloro-5-iodophenyl)thio)-9H-purin-6-amine(56, HJP-VI-6)

Yield, 10.8 mg (91.5%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.26 (s,1H), 7.72 (t, J=1.4 Hz, 1H), 7.70 (t, J=1.4 Hz, 1H), 7.43 (t, J=1.7 Hz,1H), 4.35 (t, J=6.9 Hz, 2H), 2.64 (t, J=6.5 Hz, 2H), 2.09-2.13 (m, 2H),1.20 (s, 9H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.7, 152.8,151.3, 144.9, 138.0, 137.6, 135.9, 133.1, 130.7, 119.6, 94.6, 53.1,41.3, 38.4, 29.1, 27.6; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H₂₅IClN₆S,531.0595; found 531.0587.

6.2.14 Synthesis of Compounds of Formula 57-87 (Scheme 14)

General Conditions:

Method A:

Boronic acid or pinacol ester (1.5-3 eq.) was added to HJP-V-149 (53, 15mg, 0.0298 mmol, 1 eq.) and NaHCO₃ (3 eq.) in a 10 mL RBF equipped witha magnetic stir bar and rubber septum. DMF (0.5 mL) was added and thereaction mixture was evacuated and back filled with nitrogen. This wasrepeated four times then nitrogen was bubbled through the reactionmixture for 10 min. Then H₂O (0.1 mL) and PdCl₂(PPh₃)₂ (10-20 mol %)were added and the reaction mixture was heated under nitrogen at 90° C.for 2-24 h. Solvent was removed under reduced pressure and the resultingresidue was purified by preparatory TLC to yield desired compounds57-63, 65-73, 81-85, 87.

Method B:

A mixture of HJP-V-149 (15 mg, 0.0298 mmol, 1 eq.), (n-Bu)₃SnR (4 eq.),LiCl (2 eq.) and Pd(PPh₃)₄ (10-20 mol %) in DMF (1 mL) in a 10 mL RBFequipped with a magnetic stir bar and rubber septum was evacuated andback filled with nitrogen. This was repeated four times then thereaction mixture was heated under nitrogen at 90-100° C. for 18 h.Solvent was removed under reduced pressure and the resulting residue waspurified by preparatory TLC to yield compounds 74 (HJP-VI-49), and 86(HJP-VI-78).

Method C:

To a solution of HJP-V-149 (15 mg, 0.0298 mmol, 1 eq.) in DMF (2 mL) ina sealed tube flushed with argon was added CuI (0.5 eq.), PdCl₂(PPh₃)₂(15 mol %), alkyne (2-2.5 eq.) and triethylamine (5 eq.). The reactionmixture was heated at 90-100° C. for 24 h. Solvent was removed underreduced pressure and the resulting residue was purified by preparatoryTLC to yield compounds 76 (HJP-VI-51), 77 (HJP-VI-52), 78 (HJP-VI-53),64 (HJP-VI-18), 75 (HJP-VI-50), 79 (HJP-VI-58), 80 (HJP-VI-59).

8-((5-chloro-4′-methoxy-[1,1′-biphenyl]-3-yl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(57, HJP-VI-3)

Yield, 9.6 mg (67%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.22 (s,1H), 7.65 (t, J=1.6 Hz, 1H), 7.60 (t, J=1.7 Hz, 1H), 7.52 (d, J=8.8, Hz,2H), 7.44 (t, J=1.7 Hz, 1H), 7.01 (d, J=8.8, Hz, 2H), 4.42 (t, J=6.9 Hz,2H), 3.86 (s, 3H), 3.30-3.34 (m, 1H), 3.01 (t, J=7.3 Hz, 2H), 2.27-2.31(m, 2H), 1.37 (d, J=6.5 Hz, 6H); HRMS (ESI) m/z [M+H]⁺ calcd. forC₂₄H₂₈ClN₆OS, 483.1734; found 483.1713.

8-((5-chloro-3′-methoxy-[1,1′-biphenyl]-3-yl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(58, HJP-VI-7)

Yield, 5.1 mg (35%). ¹H NMR (600 MHz, CDCl₃) δ 8.32 (s, 1H), 7.65 (t,J=1.6 Hz, 1H), 7.52 (t, J=1.6 Hz, 1H), 7.50 (t, J=1.7, Hz, 1H), 7.37 (t,J=1.7 Hz, 1H), 7.35 (t, J=7.9, Hz, 1H), 7.08 (d, J=7.6 Hz, 1H), 7.02 (s,1H), 6.92 (dd, J=8.2 and 1.9 Hz, 1H), 5.72 (br s, 2H), 4.35 (t, J=6.9Hz, 2H), 3.84 (s, 3H), 2.74-2.79 (m, 1H), 2.58 (t, J=6.7 Hz, 2H),2.01-2.06 (m, 2H), 1.07 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ160.0, 154.6, 153.1, 151.7, 144.9, 144.0, 140.0, 135.6, 132.9, 130.1,129.0, 127.6, 127.4, 120.1, 119.5, 113.8, 112.8, 55.4, 49.1, 43.4, 41.5,29.6, 22.3; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₄H₂₈ClN₆OS, 483.1734;found 483.1721.

8-((5-chloro-3′-nitro-[1,1′-biphenyl]-3-yl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(59, HJP-VI-8)

Yield, 11.8 mg (78%). ¹H NMR (600 MHz, CDCl₃) δ 8.40 (s, 1H), 8.33 (s,1H), 8.25 (d, J=8.0 Hz, 1H), 7.85 (d, J=7.9 Hz, 1H), 7.65 (s, 1H), 7.63(t, J=8.0 Hz, 1H), 7.54 (s, 1H), 7.46 (s, 1H), 5.82 (br s, 2H), 4.36 (t,J=6.9 Hz, 2H), 2.70-2.74 (m, 1H), 2.58 (t, J=6.8, Hz, 2H), 1.98-2.04 (m,2H), 1.03 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.6, 153.2,151.6, 148.7, 144.5, 141.4, 140.3, 136.0, 133.9, 132.9, 130.1, 130.0,127.5, 127.2, 123.2, 122.1, 120.1, 48.8, 43.7, 41.7, 30.2, 22.8; HRMS(ESI) m/z [M+H]⁺ calcd. for C₂₃H₂₅ClN₇O₂S, 498.1479; found 498.1483.

8-((5-chloro-3′-(trifluoromethyl)-[1,1′-biphenyl]-3-yl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(60, HJP-VI-9)

Yield, 9.0 mg (59%). ¹H NMR (600 MHz, CDCl₃) δ 8.33 (s, 1H), 7.76 (s,1H), 7.69 (d, J=7.7 Hz, 1H), 7.65 (d, J=7.7 Hz, 1H), 7.55-7.59 (m, 2H),7.51 (t, J=1.7 Hz, 1H), 7.42 (t, J=1.7 Hz, 1H), 5.74 (br s, 2H), 4.35(t, J=6.9 Hz, 2H), 2.72-2.77 (m, 1H), 2.58 (t, J=6.7 Hz, 2H), 1.99-2.05(m, 2H), 1.04 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.6,153.2, 151.7, 144.7, 142.6, 139.4, 135.8, 133.6, 131.5 (q, J_(C-F)=32.1Hz), 130.4, 129.62, 129.61, 127.5, 127.3, 125.1 (q, J_(C-F)=3.5 Hz),123.94 (q, J_(C-F)=3.8 Hz), 123.87 (q, J_(C-F)=270.8 Hz) 120.1, 48.9,43.6, 41.6, 29.9, 22.5; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₄H₂₅ClN₆F₃S,521.1502; found 521.1513.

8-((3-chloro-5-(thiophen-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(61, HJP-VI-10)

Yield, 11.0 mg (80%). ¹H NMR (600 MHz, CDCl₃) δ 8.33 (s, 1H), 7.55 (t,J=1.5 Hz, 1H), 7.51 (t, J=1.7 Hz, 1H), 7.33 (d, J=5.1 Hz, 1H), 7.29 (d,J=3.6 Hz, 1H), 7.27 (t, J=1.7 Hz, 1H), 7.07 (t, J=5.0 Hz, 1H), 5.81 (brs, 2H), 4.34 (t, J=6.9 Hz, 2H), 2.71-2.75 (m, 1H), 2.56 (t, J=6.7 Hz,2H), 1.96-2.03 (m, 2H), 1.04 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃)δ 154.6, 153.2, 151.6, 144.6, 141.3, 137.1, 135.7, 133.5, 128.7, 128.3,126.4, 125.9, 125.7, 124.7, 120.1, 48.8, 43.7, 41.7, 30.0, 22.6; HRMS(ESI) m/z [M+H]⁺ calcd. for C₂₁H₂₄ClN₆S₂, 459.1192; found 459.1202.

8-((3-chloro-5-(prop-1-en-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(62, HJP-VI-12)

Yield, 9.2 mg (75%). ¹H NMR (600 MHz, CDCl₃) δ 8.32 (s, 1H), 7.39 (t,J=1.6 Hz, 1H), 7.33 (t, J=1.7 Hz, 1H), 7.26 (t, J=1.8 Hz, 1H), 5.33-5.36(m, 1H), 5.10-5.15 (m, 1H), 6.16 (br s, 2H), 4.31 (t, J=7.1 Hz, 2H),2.65-2.72 (m, 1H), 2.53 (t, J=6.8 Hz, 2H), 2.07 (s, 3H), 1.92-1.98 (m,2H), 1.02 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.8, 153.2,151.6, 144.6, 144.1, 141.1, 135.2, 132.8, 128.8, 125.8, 125.7, 120.1,114.9, 48.7, 43.8, 41.8, 30.3, 22.9, 21.5; HRMS (ESI) m/z [M+H]⁺ calcd.for C₂₀H₂₆ClN₆S, 417.1628; found 417.1630.

8-((3-chloro-5-(3-methylbut-2-en-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(63, HJP-VI-14)

Yield, 9.6 mg (72%). ¹H NMR (600 MHz, CDCl₃ δ 8.33 (s, 1H), 7.22 (t,J=1.8 Hz, 1H), 7.05 (t, J=1.5 Hz, 1H), 7.01 (t, J=1.6 Hz, 1H), 5.76 (brs, 2H), 4.30 (t, J=7.0 Hz, 2H), 2.68-2.72 (m, 1H), 2.53 (t, J=6.7 Hz,2H), 1.92-1.98 (m, 2H), 1.89 (s, 3H), 1.76 (s, 3H), 1.54 (s, 3H), 1.03(d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.6, 153.1, 151.6,148.1, 144.9, 134.7, 132.4, 129.5, 128.7, 128.5, 127.8, 127.3, 120.1,48.7, 43.7, 41.7, 30.1, 22.8, 22.1, 20.6, 20.5; HRMS (ESI) m/z [M+H]⁺calcd. for C₂₂H3₀ClN₆S, 445.1941; found 445.1939.

8-((3-chloro-5-ethynylphenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(64, HJP-VI-18)

Yield, 9 mg (76%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.26 (s, 1H),7.46 (t, J=1.5 Hz, 1H), 7.45 (t, J=1.5 Hz, 1H), 7.43 (t, J=1.7 Hz, 1H),4.31 (t, J=6.9 Hz, 2H), 3.22 (s, 1H), 2.73-2.79 (m, 1H), 2.56 (t, J=6.8Hz, 2H), 1.98-2.06 (m, 2H), 1.08 (d, J=6.3 Hz, 6H); ¹³C NMR (150 MHz,CDCl₃+5 drops CD₃OD) δ 154.6, 152.9, 151.4, 144.9, 135.4, 132.9, 132.3,132.2, 131.6, 125.3, 119.7, 80.9, 80.3, 48.9, 43.2, 41.5, 29.5, 22.1;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H2₂ClN₆S, 401.1315; found 401.1324.

8-((3-chloro-5-(1H-pyrrol-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(65, HJP-VI-28)

Yield, 7.4 mg (68%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.24 (s,1H), 7.62 (s, 1H), 7.54 (s, 1H), 7.25 (s, 1H), 6.87-6.90 (m, 1H), 6.53(d, J=3.4 Hz, 1H), 6.25 (t, J=3.1 Hz, 1H), 4.31 (t, J=7.1 Hz, 2H),2.75-2.78 (m, 1H), 2.59 (t, J=6.9 Hz, 2H), 1.96-2.01 (m, 2H), 1.07 (d,J=6.3 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.5, 152.9,151.2, 146.1, 136.5, 135.6, 131.5, 129.3, 128.5, 125.6, 124.9, 120.7,119.4, 110.0, 107.6, 49.0, 43.4, 41.5, 29.2, 21.9; HRMS (ESI) m/z [M+H]⁺calcd. for C₂₁H₂₅ClN₇S, 442.1581; found 442.1592.

8-((3-chloro-5-(1H-pyrazol-5-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(66, HJP-VI-29)

Yield, 8.2 mg (94%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.33 (s,1H), 7.77 (s, 1H), 7.60 (t, J=1.4 Hz, 1H), 7.56 (d, J=2.2 Hz, 1H), 7.35(t, J=1.7 Hz, 1H) 6.69 (br s, 2H), 6.53 (d, J=2.2 Hz, 1H), 4.33 (t,J=7.0 Hz, 2H), 2.70-2.75 (m, 1H), 2.58 (t, J=6.8 Hz, 2H), 1.94-1.98 (m,2H), 1.03 (d, J=6.3 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ154.9, 153.2, 151.4, 147.6, 144.4, 135.8, 133.3, 131.3, 129.1, 125.8,125.7, 120.0, 102.9, 48.8, 43.6, 41.8, 30.5, 22.5; HRMS (ESI) m/z [M+H]⁺calcd. for C₂₀H2₄ClN₈S, 443.1533; found 443.1522.

8-((3-chloro-5-(furan-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(67, HJP-VI-30)

Yield, 9.2 mg (69%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.26 (s,1H), 7.66 (t, J=1.4 Hz, 1H), 7.63 (t, J=1.6 Hz, 1H), 7.49 (d, J=1.3 Hz,1H), 6.72 (d, J=3.4 Hz, 1H), 6.48-6.50 (m, 1H), 4.33 (t, J=6.9 Hz, 2H),2.78-2.81 (m, 1H), 2.58 (t, J=6.8 Hz, 2H), 2.02-2.07 (m, 2H), 1.09 (d,J=6.3 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.6, 153.7,152.8, 151.4, 149.4, 145.9, 135.8, 134.0, 131.8, 129.2, 124.6, 123.7,119.5, 108.6, 108.4, 49.9, 43.3, 41.5, 29.3, 22.0, 13.7; HRMS (ESI) m/z[M+H]⁺ calcd. for C₂₂H₂₆ClN₆OS, 457.1577; found 457.1578.

8-((3-chloro-5-vinylphenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(68, HJP-VI-31)

Yield, 6.7 mg (56%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.25 (s,1H), 7.41 (s, 1H), 7.39 (s, 1H), 7.33 (s, 1H), 6.58-6.65 (m, 1H), 5.79(d, J=17.5 Hz, 1H), 5.38 (d, J=10.9 Hz, 1H), 4.32 (t, J=6.8 Hz, 2H),2.81-2.86 (m, 1H), 2.61 (t, J=6.8 Hz, 2H), 2.03-2.05 (m, 2H), 1.13 (d,J=6.3 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.6, 152.8,151.4, 145.9, 140.8, 135.7, 134.4, 131.5, 130.5, 127.9, 126.7, 119.5,117.2, 49.3, 48.7, 43.0, 41.3, 28.9, 21.7; HRMS (ESI) m/z [M+H]⁺ calcd.for C₁₉H2₄ClN₆S, 403.1472; found 403.1461.

8-((3-chloro-5-(5-methylfuran-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(69, HJP-VI-38)

Yield, 10.1 mg (74%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.24 (s,1H), 7.63 (s, 1H), 7.59 (s, 1H), 7.25 (s, 1H), 6.61 (d, J=3.2 Hz, 1H),6.08 (d, J=3.1 Hz, 1H), 4.32 (t, J=6.9 Hz, 2H), 2.74-2.78 (m, 1H), 2.57(t, J=6.8 Hz, 2H), 2.36 (s, 3H), 1.98-2.04 (m, 2H), 1.07 (d, J=6.3 Hz,6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.6, 152.8, 151.4,151.1, 145.6, 143.4, 135.8, 133.7, 132.1, 129.6, 124.9, 124.1, 119.6,112.1, 107.5, 49.1, 43.1, 41.4, 29.2, 21.9; HRMS (ESI) m/z [M+H]⁺ calcd.for C₂₁H₂₄ClN₆OS, 443.1421; found 443.1403.

8-((3-chloro-5-(furan-3-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(70, HJP-VI-39)

Yield, 8.8 mg (67%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.24 (s,1H), 7.79 (s, 1H), 7.54 (s, 1H), 7.50 (s, 1H), 7.48 (s, 1H), 7.34 (s,1H), 6.67 (s, 1H), 4.38 (t, J=6.7 Hz, 2H), 2.99-3.03 (m, 1H), 2.73 (t,J=6.7 Hz, 2H), 2.16-2.21 (m, 2H), 1.24 (d, J=6.4 Hz, 6H); ¹³C NMR (150MHz, CDCl₃+5 drops CD₃OD) δ 154.6, 152.7, 151.4, 146.1, 144.4, 139.7,135.9, 131.4, 129.9, 127.6, 126.6, 124.3, 119.4, 108.5, 50.0, 42.4,40.9, 31.6, 20.6; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₁H2₄ClN₆OS,443.1421; found 443.1410.

8-((3-chloro-5-(1H-pyrrol-3-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(71, HJP-VI-44)

Yield, 4.4 mg (35%). ¹H NMR (600 MHz, CDCl₃) δ 8.32 (s, 1H), 7.45 (t,J=1.5 Hz, 1H), 7.43 (t, J=1.5 Hz, 1H), 7.16 (t, J=1.7 Hz, 1H), 7.05-7.06(m, 1H), 6.81-6.83 (m, 1H), 6.44-6.46 (m, 1H), 5.68 (br s, 2H), 4.33 (t,J=6.9 Hz, 2H), 2.72-2.76 (m, 1H), 2.56 (t, J=6.7 Hz, 2H), 1.95-2.02 (m,2H), 1.04 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.5, 153.1,151.7, 145.2, 138.9, 135.4, 132.6, 126.9, 125.5, 125.1, 122.6, 120.0,119.5, 115.5, 106.5, 48.9, 43.6, 41.6, 28.7, 22.5; HRMS (ESI) m/z [M+H]⁺calcd. for C₂₁H₂₅ClN₇S, 442.1581; found 442.1586.

8-((3-chloro-5-(1H-pyrazol-4-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(72, HJP-VI-46)

Yield, 8.9 mg (67%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.24 (s,1H), 7.83 (s, 2H), 7.54 (s, 1H), 7.49 (s, 1H), 7.29 (s, 1H), 4.33 (t,J=6.8 Hz, 2H), 2.82-2.87 (m, 1H), 2.64 (t, J=6.7 Hz, 2H), 2.05-2.11 (m,2H), 1.12 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ154.7, 154.6, 152.7, 151.3, 145.9, 136.1, 135.8, 131.7, 129.2, 127.2,126.2, 119.9, 119.5, 119.4, 49.3, 43.0, 41.3, 28.9, 21.6; HRMS (ESI) m/z[M+H]⁺ calcd. for C₂₀H2₄ClN₈S, 443.1533; found 443.1536.

8-((3-chloro-5-(1-methyl-1H-pyrazol-3-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(73, HJP-VI-47)

Yield, 10.8 mg (79%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.27 (s,1H), 7.67 (s, 1H), 7.58 (s, 1H), 7.53-7.55 (m, 1H), 7.31 (s, 1H), 6.39(s, 1H), 4.33 (t, J=6.7 Hz, 2H), 3.27-3.32 (m, 1H), 3.02 (t, J=7.3 Hz,2H), 2.30-2.33 (m, 2H), 1.33 (d, J=6.5 Hz, 6H); ¹³C NMR (150 MHz,CDCl₃+5 drops CD₃OD) δ 150.5, 150.4, 150.1, 144.7, 140.9, 138.9, 136.0,133.9, 133.4, 132.2, 130.4, 129.1, 118.9, 107.1, 50.8, 41.9, 41.3, 37.5,26.1, 18.9; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₁H₂₆ClN₈S, 457.1690;found 457.1685.

8-((3-chloro-5-(oxazol-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(74, HJP-VI-49)

Yield, 7.6 mg (57%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.24 (s,1H), 8.09 (s, 1H), 8.04 (s, 1H), 7.77 (s, 1H), 7.55 (s, 1H), 7.30 (s,1H), 4.39 (t, J=6.6 Hz, 2H), 3.01-3.06 (m, 1H), 3.74 (t, J=6.7 Hz, 2H),2.18-2.24 (m, 2H), 1.25 (d, J=6.4 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5drops CD₃OD) δ 159.5, 154.7, 152.7, 151.4, 145.4, 139.8, 136.1, 132.9,132.5, 130.1, 128.8, 127.5, 126.8, 119.6, 50.1, 42.3, 40.9, 27.8, 20.5;HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₀H₂₃ClN₇OS, 444.1373; found 444.1362.

8-((3-chloro-5-(prop-1-yn-1-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(75, HJP-VI-50)

Yield, 4.8 mg (40%). ¹H NMR (600 MHz, CD₃OD) δ 8.31 (s, 1H), 7.54 (t,J=1.8 Hz, 1H), 7.47 (t, J=1.4 Hz, 1H), 7.42 (t, J=1.6 Hz, 1H), 4.43 (t,J=6.9 Hz, 2H), 3.30-3.34 (m, 1H), 3.06 (t, J=6.9 Hz, 2H), 2.18-2.21 (m,2H), 2.03 (s, 3H), 1.31 (d, J=6.5 Hz, 6H); ¹³C NMR (150 MHz, CD₃OD) δ153.6, 152.2, 149.7, 148.8, 136.4, 134.2, 132.9, 132.8, 132.0, 128.8,120.7, 90.5, 78.1, 52.2, 43.3, 40.4, 27.7, 19.3, 3.8; HRMS (ESI) m/z[M+H]⁺ calcd. for C₂₀H2₄ClN₆S, 415.1472; found 415.1474.

8-((3-chloro-5-(3-methylbut-1-yn-1-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(76, HJP-VI-51)

Yield, 9.2 mg (70%). ¹H NMR (600 MHz, CDCl₃) δ 8.33 (s, 1H), 7.30 (t,J=1.6 Hz, 1H), 7.29 (t, J=1.4 Hz, 1H), 7.27 (m, 1H), 5.84 (br s, 2H),4.30 (t, J=7.1 Hz, 2H), 2.68-2.75 (m, 2H), 2.53 (t, J=6.8 Hz, 2H),1.93-1.96 (m, 2H), 1.22 (d, J=6.9 Hz, 6H), 1.03 (d, J=6.2 Hz, 6H); ¹³CNMR (150 MHz, CDCl₃) δ 154.7, 153.3, 151.6, 144.3, 134.9, 132.9, 131.3,131.2, 129.1, 126.9, 120.2, 98.8, 77.6, 48.7, 43.8, 41.8, 30.2, 22.8,22.7, 21.1; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₂H₂₈ClN₆S, 443.1785;found 443.1774.

8-((3-chloro-5-(cyclopropylethynyl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(77, HJP-VI-52)

Yield, 8.4 mg (64%). ¹H NMR (600 MHz, CDCl₃) δ 8.34 (s, 1H), 7.25-7.27(m, 2H), 7.24 (t, J=1.4 Hz, 1H), 5.85 (br s, 2H), 4.29 (t, J=7.0 Hz,2H), 2.68-2.72 (m, 2H), 2.53 (t, J=6.8 Hz, 2H), 1.93-1.96 (m, 2H),1.40-1.42 (m, 1H), 1.02 (d, J=6.2 Hz, 6H), 0.85-0.89 (m, 2H), 0.78-0.81(m, 2H); ¹³C NMR (150 MHz, CDCl₃) δ 154.6, 153.2, 151.5, 144.1, 134.8,132.9, 131.1, 131.0, 128.8, 126.8, 120.1, 96.5, 73.5, 48.6, 43.7, 41.7,30.1, 22.7, 8.6; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₂H₂₆ClN₆S, 441.1628;found 441.1628.

8-((3-chloro-5-(3,3-dimethylbut-1-yn-1-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(78, HJP-VI-53)

Yield, 9.1 mg (67%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.26 (s,1H), 7.35-7.37 (m, 2H), 7.32 (t, J=1.7 Hz, 1H), 4.29 (t, J=6.9 Hz, 2H),2.65-2.67 (m, 1H), 2.54 (t, J=6.8 Hz, 2H), 1.96-1.99 (m, 2H), 1.29 (s,9H), 1.06 (d, J=6.3 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ154.6, 152.9, 151.4, 145.3, 135.1, 132.6, 131.9, 131.6, 130.3, 127.3,119.6, 101.8, 76.8, 48.8, 43.3, 41.5, 30.7, 29.6, 28.0, 22.3; HRMS (ESI)m/z [M+H]⁺ calcd. for C₂₃H₃₀ClN₆S, 457.1941; found 457.1945.

3-(3-((6-amino-9-(3-(isopropylamino)propyl)-9H-purin-8-yl)thio)-5-chlorophenyl)prop-2-yn-1-ol(79, HJP-VI-58)

Yield, 4.5 mg (35%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.26 (s,1H), 7.41 (t, J=1.8 Hz, 1H), 7.37-7.38 (m, 2H), 4.31 (t, J=6.9 Hz, 2H),2.81-2.84 (m, 1H), 2.61 (t, J=6.8 Hz, 2H), 2.01-2.06 (m, 2H), 1.12 (d,J=6.3 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.6, 152.9,151.3, 145.1, 135.3, 132.6, 131.9, 131.7, 131.1, 126.0, 119.6, 90.9,82.3, 50.8, 49.2, 43.0, 41.4, 29.0, 21.7; HRMS (ESI) m/z [M+H]⁺ calcd.for C₂₀H2₄ClN₆OS, 431.1421; found 431.1431.

4-(3-((6-amino-9-(3-(isopropylamino)propyl)-9H-purin-8-yl)thio)-5-chlorophenyl)but-3-yn-2-ol(80, HJP-VI-59)

Yield, 5.6 mg (42%). ¹H NMR (600 MHz, CD₃OD) δ 8.35 (s, 1H), 7.60 (s,1H), 7.52 (s, 1H), 7.48-7.49 (m, 1H), 4.64-4.68 (m, 1H), 4.43 (t, J=6.9Hz, 2H), 3.30-3.31 (m, 1H), 3.07 (t, J=7.7 Hz, 2H), 2.19-2.21 (m, 2H),1.46 (d, J=6.7 Hz, 3H), 1.31 (d, J=6.54 Hz, 6H); ¹³C NMR (150 MHz,CD₃OD) δ 153.7, 152.2, 149.8, 148.6, 136.5, 134.2, 133.2, 132.9, 132.7,127.6, 95.7, 81.4, 58.9, 49.6, 43.3, 42.1, 27.7, 24.5, 19.3; HRMS (ESI)m/z [M+H]⁺ calcd. for C₂₀H2₄ClN₆OS, 431.1421; found 431.1431.

8-((3-chloro-5-(2-methylprop-1-en-1-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(81, HJP-VI-62)

Yield, 5.4 mg (42%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.28 (s,1H), 7.38 (s, 1H), 7.28-7.31 (m, 2H), 6.18 (s, 1H), 4.38 (t, J=6.7 Hz,2H), 3.27-3.29 (m, 1H), 2.98 (t, J=7.02 Hz, 2H), 2.25-2.29 (m, 2H), 1.92(s, 3H), 1.85 (s, 3H), 1.33 (d, J=6.5 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5drops CD₃OD) δ 151.4, 150.8, 149.9, 146.9, 142.3, 139.5, 135.2, 132.1,130.3, 130.2, 128.3, 122.6, 50.9, 41.9, 41.0, 26.9, 26.2, 19.5, 18.9;HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₁H₂₈ClN₆S, 431.1785; found 431.1794.

(E)-8-((3-chloro-5-(prop-1-en-1-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(82, HJP-VI-63)

Yield, 7.3 mg (59%). ¹H NMR (600 MHz, CDCl₃) δ 8.32 (s, 1H), 7.21 (t,J=1.6 Hz, 1H), 7.19 (t, J=1.5 Hz, 1H), 7.18 (t, J=1.7 Hz, 1H), 6.22-6.25(m, 2H), 6.17 (br s, 2H), 4.64-4.68 (m, 1H), 4.29 (t, J=7.1 Hz, 2H),2.67-2.71 (m, 1H), 2.52 (t, J=6.8 Hz, 2H), 1.91-1.97 (m, 2H), 1.85 (d,J=4.9 Hz, 3H), 1.02 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.8,153.2, 151.6, 144.5, 140.8, 135.3, 132.9, 129.2, 128.8, 127.9, 125.9,125.5, 120.1, 48.7, 43.8, 41.8, 30.2, 22.8, 18.5; HRMS (ESI) m/z [M+H]⁺calcd. for C₂₀H₂₆ClN₆S, 417.1628; found 417.1627.

(E)-8-((3-chloro-5-(2-cyclopropylvinyl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(83, HJP-VI-64)

Yield, 8.2 mg (62%). ¹H NMR (600 MHz, CDCl₃) δ 8.25 (s, 1H), 7.28-7.29(m, 1H), 7.25-7.26 (m, 1H), 7.21 (t, J=1.7 Hz, 1H), 6.35 (d, J=15.7 Hz,1H), 5.72-5.78 (m, 1H), 4.28 (t, J=7.0 Hz, 2H), 2.68-2.72 (m, 1H), 2.52(t, J=6.9 Hz, 2H), 1.95-1.98 (m, 2H), 1.54-1.57 (m, 1H), 1.22 (s, 1H),1.05 (d, J=6.3 Hz, 6H), 0.84-0.88 (m, 2H), 0.52-0.55 (m, 2H); ¹³C NMR(150 MHz, CDCl₃) δ 154.5, 152.8, 151.4, 145.9, 141.1, 135.8, 135.5,131.5, 128.9, 127.2, 125.9, 124.8, 119.5, 48.7, 43.4, 41.5, 29.7, 24.6,22.4, 14.8, 7.67; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₂H₂₈ClN₆S,443.1785; found 443.1775.

8-((3-chloro-5-(3,3,3-trifluoroprop-1-en-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(84, HJP-VI-70)

Yield, 8.8 mg (61%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.23 (s,1H), 7.52-7.53 (m, 1H), 7.50 (s, 1H), 7.47 (s, 1H), 6.08 (s, 1H), 5.90(s, 1H), 4.40 (t, J=6.7 Hz, 2H), 3.18-3.23 (m, 1H), 2.86 (t, J=6.8 Hz,2H), 2.25-2.31 (m, 2H), 1.35 (d, J=6.5 Hz, 6H); ¹³C NMR (150 MHz,CDCl₃+5 drops CD₃OD) δ 154.8, 152.7, 151.3, 145.9, 136.7 (q,J_(C-F)=30.9 Hz), 136.6, 135.9, 132.1, 131.6, 129.2, 128.4, 122.8 (q,J_(C-F)=272.2 Hz), 123.1 (q, J_(C-F)=5.5 Hz), 119.5, 50.9, 41.9, 40.6,26.9, 19.5; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₀H₂₃F₃ClN₆S, 471.1346;found 471.1338.

(E)-8-((3-chloro-5-(3,3-dimethylbut-1-en-1-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(85, HJP-VI-72)

Yield, 9.2 mg (67%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.22 (s,1H), 7.37-7.39 (m, 2H), 7.29-7.33 (m, 1H), 6.32 (d, J=16.1 Hz, 1H), 6.21(d, J=16.1 Hz, 1H), 4.37 (t, J=6.3 Hz, 2H), 3.11-3.14 (m, 1H), 2.79 (t,J=6.4 Hz, 2H), 2.21-2.25 (m, 2H), 1.31 (d, J=6.2 Hz, 6H), 1.11 (s, 9H);¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.7, 152.5, 151.3, 146.5,145.4, 141.6, 135.6, 130.7, 129.8, 126.8, 122.4, 119.4, 50.5, 42.1,40.7, 33.7, 29.33, 27.2, 19.9; HRMS (ESI) m/z [M+H]⁺ calcd. forC₂₃H₃₂ClN₆S, 459.2098; found 459.2083.

(Z)-8-((3-chloro-5-(prop-1-en-1-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(86, HJP-VI-78)

Yield, 5.3 mg (43%). ¹H NMR (600 MHz, CDCl₃) δ 8.32 (s, 1H), 7.18-7.26(m, 3H), 6.25-6.31 (m, 1H), 5.83-5.89 (m, 1H), 5.79 (br s, 2H), 4.33 (t,J=6.8 Hz, 2H), 2.74-2.78 (m, 1H), 2.57 (t, J=6.7 Hz, 2H), 1.97-2.04 (m,2H), 1.82 (dd, J=7.2 and 1.7 Hz, 3H), 1.08 (d, J=6.3 Hz, 6H); ¹³C NMR(150 MHz, CDCl₃) δ 154.6, 153.1, 151.6, 145.0, 140.4, 134.9, 132.4,129.7, 128.9, 128.7, 127.9, 127.6, 120.1, 49.0, 43.5, 41.5, 29.7, 22.4,18.5; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₀H₂₆ClN₆S, 417.1628; found417.1634.

(E)-8-((3-(but-2-en-2-yl)-5-chlorophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(87, HJP-VI-79)

Yield, 9.4 mg (73%). ¹H NMR (600 MHz, CDCl₃) δ 8.33 (s, 1H), 7.29-7.30(m, 1H), 7.26-7.27 (m, 1H), 7.21-7.22 (m, 1H), 5.84-5.89 (m, 1H), 5.82(br s, 2H), 4.30 (t, J=7.0 Hz, 2H), 2.68-2.73 (m, 1H), 2.54 (t, J=6.8Hz, 2H), 1.93-1.98 (m, 5H), 1.77 (d, J=6.8 Hz, 3H), 1.02 (d, J=6.2 Hz,6H); ¹³C NMR (150 MHz, CDCl₃) δ 154.6, 153.1, 151.6, 145.9, 135.0,133.5, 132.3, 128.0, 126.1, 125.8, 125.2, 120.1, 48.8, 43.7, 41.7, 30.1,22.8, 15.3, 14.4; HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₁H₂₈ClN₆S,431.1785; found 431.1782.

6.2.15 Synthesis of Compounds of Formula 91-95 (Scheme 15)

8-((4-chloro-2-nitrophenyl)thio)-9H-purin-6-amine (89)

8-Mercaptoadenine (3.6 mmol), neocuproine hydrate (0.36 mmol), CuI (0.36mmol), NaO-t-Bu (7.2 mmol), 4-chloro-1-iodo-2-nitrobenzene (10.8 mmol),and anhydrous DMF (24 mL) were taken in a round bottom flask flushedwith nitrogen. The flask was sealed with Teflon tape, heated at 110° C.,and magnetically stirred for 18 h under nitrogen. Solvent was removedunder reduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5). Obtained as a yellow solid in 85% yield.MS (ESI): m/z 332.8 [M+H]⁺.

9-(3-bromopropyl)-8-((4-chloro-2-nitrophenyl)thio)-9H-purin-6-amine (90)

8-Arylsulfanyl adenine (89, 1.21 mmol) was dissolved in DMF (15 mL) andCs₂CO₃ (1.45 mmol) and 1,3-dibromopropane (2.42 mmol) were added and themixture was stirred under nitrogen at for 2-4 h. Solvent was removedunder reduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5) to afford desired compound 90. Obtained asa solid in 35% yield. MS (ESI): m/z 442.9 [M+H]⁺.

8-((4-chloro-2-nitrophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(91, HJP-VI-32)

A mixture of 90 (600 mg, 1.357 mmol) and amine (67.9 mmol, 50 equiv.) inDMF (8 mL) under nitrogen protection was stirred at room temperature for20 hrs. Following solvent removal, the crude material was purified bycolumn chromatography (CH₂Cl₂:CH₃OH—NH₃ (7N), 100:1 to 20:1) to afforddesired product 91. Yield, 510 mg (85%). ¹H NMR (600 MHz, CDCl₃+5 dropsCD₃OD) δ 8.35 (s, 1H), 8.30 (d, J=2.2 Hz, 1H), 7.43 (dd, J=8.7 and 2.2Hz, 1H), 6.81 (d, J=8.7 Hz, 1H), 4.29 (t, J=6.9 Hz, 2H), 2.67-2.73 (m,1H), 2.52 (t, J=6.8 Hz, 2H), 1.93-1.98 (m, 2H), 1.03 (d, J=6.3 Hz, 6H);¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.3, 153.9, 151.3, 145.8,142.0, 134.5, 133.3, 131.9, 129.7, 126.2, 120.4, 48.7, 43.3, 41.9, 30.1,22.3; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₂₁ClN₇OS, 422.1166; found422.1170.

8-((2-amino-4-chlorophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(92, HJP-VI-34)

A mixture of 91 (510 mg, 1.21 mmol) and iron powder (250 mg.) in aceticacid (6 mL) was stirred at room temperature for 4 hrs. On completionreaction was neutralized by adding solid Na₂CO₃ at 0° C. and washed withEtOAc (75 ml×3). Following drying over MgSO₄ and solvent removal, thecrude material was purified by column chromatography (CH₂Cl₂:CH₃OH—NH₃(7N), 50:1 to 15:1) to afford desired product 92. Yield, 426.3 mg (90%).¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.16 (s, 1H), 7.39 (d, J=8.3 Hz,1H), 6.84 (d, J=2.2 Hz, 1H), 6.73 (dd, J=8.3 and 2.2 Hz, 1H), 4.36 (t,J=6.7 Hz, 2H), 3.40-3.41 (m, 1H), 2.83 (t, J=6.8 Hz, 2H), 2.26-2.32 (m,2H), 1.34 (d, J=6.5 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ154.2, 151.9, 151.4, 150.5, 147.3, 138.3, 138.1, 118.9, 115.7, 107.0,74.4, 50.6, 42.1, 40.4, 31.1, 19.8; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H2₃ClN₇S, 392.1424; found 392.1419.

8-((4-chloro-2-iodophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(93, HJP-VI-36)

A mixture of 92 (426 mg, 1.09 mmol), NaNO₂ (1.2 equiv.) and potassiumiodide (2 equiv.) in acetic acid (5 mL) was stirred at room temperaturefor 4 hrs. On completion reaction was neutralized by adding solid Na₂CO₃at 0° C. and washed with EtOAc (75 ml×3). Following drying over MgSO₄and solvent removal, the crude material was purified by columnchromatography (CH₂Cl₂:CH₃OH—NH₃ (7N), 80:1 to 20:1) to afford desiredproduct 93. Yield, 436.4 mg (79%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD)δ 8.35 (s, 1H), 7.87 (d, J=2.2 Hz, 1H), 7.23 (dd, J=8.5 and 2.2 Hz, 1H),7.07 (d, J=2.2 Hz, 1H), 5.87 (br s, 2H), 4.30 (t, J=6.9 Hz, 2H),2.68-2.72 (m, 1H), 2.56 (t, J=6.8 Hz, 2H), 1.95-1.99 (m, 2H), 1.03 (d,J=6.2 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 154.7, 153.3,151.6, 144.8, 139.3, 135.9, 134.1, 131.1, 129.5, 120.4, 99.8, 48.7,43.8, 41.9, 30.3, 22.9; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₂₁IClN₆S,503.0282; found 503.0277.

General Conditions. Method A

Boronic acid or pinacol ester (1.5-3 eq.) was added to HJP-VI-36 (93, 15mg, 0.0298 mmol, 1 eq.) and NaHCO₃ (3 eq.) in a 10 mL RBF equipped witha magnetic stir bar and rubber septum. DMF (0.5 mL) was added and thereaction mixture was evacuated and back filled with nitrogen. This wasrepeated four times then nitrogen was bubbled through the reactionmixture for 10 min. Then H₂O (0.1 mL) and PdCl₂(PPh₃)₂ (10-20 mol %)were added and the reaction mixture was heated under nitrogen at 90° C.for 2-24 h. Solvent was removed under reduced pressure and the resultingresidue was purified by preparatory TLC to yield compounds HJP-VI-42(94) and HJP-VI-43 (95).

8-((4-chloro-2-(1H-pyrrol-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(94, HJP-VI-42)

Yield, 2.3 mg (24%). ¹H NMR (600 MHz, CD₃OD) δ 8.29 (s, 1H), 7.57 (d,J=2.3 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.31 (dd, J=8.4 and 2.3 Hz, 1H),6.85-6.88 (m, 1H), 6.42-6.44 (m, 1H), 6.13-6.16 (m, 1H), 4.29 (t, J=6.9Hz, 2H), 3.28-3.32 (m, 1H), 2.99 (t, J=6.2 Hz, 2H), 2.10-2.16 (m, 2H),1.29 (d, J=6.5 Hz, 6H); ¹³C NMR (150 MHz, CD₃OD) δ 152.8, 152.0, 150.9,148.7, 139.9, 137.5, 136.9, 129.2, 128.5, 126.2, 121.3, 121.2, 120.4,111.7, 110.0, 49.6, 43.3, 41.9, 27.6, 19.3; HRMS (ESI) m/z [M+H]⁺ calcd.for C₂₁H₂₅ClN₇S, 442.1581; found 442.1573.

8-((4-chloro-2-(1H-pyrazol-5-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(95, HJP-VI-43)

Yield, 10.4 mg (28%). H NMR (600 MHz, CD₃OD) δ 8.28 (s, 1H), 7.97 (s,1H), 7.69 (d, J=2.2 Hz, 1H), 7.68 (d, J=2.2 Hz, 1H), 7.46 (d, J=8.5 Hz,1H), 7.41 (dd, J=8.5 and 2.3 Hz, 1H), 4.28 (t, J=7.0 Hz, 2H), 3.27-3.31(m, 1H), 2.99 (t, J=8.0 Hz, 2H), 2.05-2.11 (m, 2H), 1.29 (d, J=6.6 Hz,6H); HRMS (ESI) m/z [M+H]⁺ calcd. for C₂₀H2₄ClN₈S, 443.1533; found443.1520.

6.2.16 Synthesis of Compounds of Formula 100-102 (Scheme 16)

8-((3,5-dichlorophenyl)thio)-9H-purine-2,6-diamine (97)

2,6-diamino-9H-purine-8-thiol (3.6 mmol), neocuproine hydrate (0.36mmol), CuI (0.36 mmol), NaO-t-Bu (7.2 mmol), 1,3-dichloro-5-iodobenzene(10.8 mmol), and anhydrous DMF (24 mL) were taken in a round bottomflask flushed with nitrogen. The flask was sealed with Teflon tape,heated at 110° C., and magnetically stirred for 20 h under nitrogen.Solvent was removed under reduced pressure and the resulting residue waschromatographed (CH₂Cl₂:MeOH:AcOH, 20:1:0.5). Obtained as a light yellowsolid in 65% yield. MS (ESI): m/z 326.9 [M+H]⁺.

8-((3,5-dichlorophenyl)thio)-2-fluoro-9H-purin-6-amine (98)

To a cooled solution (0° C.) of 97 (350 mg, 1.073 mmol) in HF/pyridine(1.5 mL) was slowly added NaNO₂ (126.2 mg, 1.73 mmol). The resultedmixture was stirred at room temperature for 1 h and then quenched bystirring for 1 h with 14 mg of CaCO₃ in CH₂Cl₂ (7.5 mL). The crudematerial was taken up in CH₂Cl₂, washed with water, and dried overanhydrous Na₂SO₄. Following solvent removal, the residue was purified ona preparative silica gel plate (CHCl₃:Hexanes:EtOAc:i-PrOH at 2:2:1:0.1)to afford 98 (180 mg, 47% yield). MS (ESI): m/z 329.80 [M+H]⁺.

9-(3-bromopropyl)-8-((3,5-dichlorophenyl)thio)-2-fluoro-9H-purin-6-amine(99)

8-Arylsulfanyl adenine (98, 0.549 mmol) was dissolved in DMF (15 mL) andCs₂CO₃ (0.659 mmol) and 1,3-dibromopropane (1.3752 mmol) were added andthe mixture was stirred under nitrogen at for 2 h. Solvent was removedunder reduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 40:1:0.5-20:1:0.5) to afford desired compound 99.Obtained as a solid in 25% yield. ¹H NMR (500 MHz, CDCl₃+5 drops CD₃OD)δ 7.28-7.34 (m, 3H), 4.31 (t, J=7.1 Hz, 2H), 3.40 (t, J=6.1 Hz, 2H),2.29-2.36 (m, 2H); MS (ESI): m/z 449.9 [M+H]⁺.

General Procedure for the Synthesis of 100-102

A mixture of 99 (12 mg, 0.0267 mmol) and amine (1.336 mmol, 50 equiv.)in DMF (1 mL) under nitrogen protection was stirred at room temperaturefor 16-24 hrs. Following solvent removal, the crude material waspurified by preparative TLC (CH₂Cl₂:CH₃OH—NH₃ (7N), 20:1 or 15:1) toafford desired product 100-102.

8-((3,5-dichlorophenyl)thio)-2-fluoro-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(100, HJP-VI-69)

Yield, 9.3 mg (81.6%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 7.30-7.36(m, 3H), 4.23 (t, J=6.9 Hz, 2H), 2.71-2.76 (m, 1H), 2.54 (t, J=6.8 Hz,2H), 1.94-1.99 (m, 2H), 1.06 (d, J=6.2 Hz, 6H); ¹³C NMR (150 MHz,CDCl₃+5 drops CD₃OD) δ 159.2 (d, J_(C-F)=211.2 Hz), 156.4 (d, J_(C-F)=20Hz), 152.7 (d, J_(C-F)=18.9 Hz), 143.9 (d, J_(C-F)=2.5 Hz), 136.1,133.7, 128.9, 128.8, 117.9 (d, J_(C-F)=3.5 Hz), 48.9, 43.4, 41.8, 29.7,22.3; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H2₀Cl₂FN₆S, 429.0831; found429.0834.

8-((3,5-dichlorophenyl)thio)-2-fluoro-9-(3-(neopentylamino)propyl)-9H-purin-6-amine(101, HJP-VI-85)

Yield, 10.2 mg (84%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 7.35 (t,J=1.7 Hz, 1H), 7.30-7.32 (m, 2H), 4.25 (t, J=7.0 Hz, 2H), 2.60 (t, J=6.8Hz, 2H), 2.31 (s, 2H), 1.96-2.01 (m, 2H), 0.93 (s, 9H); ¹³C NMR (150MHz, CDCl₃+5 drops CD₃OD) δ 159.3 (d, J_(C-F)=211.1 Hz), 156.4 (d,J_(C-F)=20.1 Hz), 152.8 (d, J_(C-F)=19.1 Hz), 144.1 (d, J_(C-F)=2.4 Hz),136.1, 133.8, 128.9, 128.8, 117.9 (d, J_(C-F)=3.5 Hz), 61.9, 47.1, 42.0,31.3, 29.2, 27.8; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₉H₂₄Cl₂FN₆S,457.1144; found 457.1152.

9-(3-(tert-butylamino)propyl)-8-((3,5-dichlorophenyl)thio)-2-fluoro-9H-purin-6-amine(102, HJP-VI-86)

Yield, 9.6 mg (80%). ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 7.36 (t,J=1.7 Hz, 1H), 7.32-7.34 (m, 2H), 4.28 (t, J=6.8 Hz, 2H), 2.66 (t, J=6.8Hz, 2H), 2.09-2.12 (m, 2H), 1.21 (s, 9H); ¹³C NMR (150 MHz, CDCl₃+5drops CD₃OD) δ 159.1 (d, J_(C-F)=211.5 Hz), 156.4 (d, J_(C-F)=20.1 Hz),152.7 (d, J_(C-F)=18.8 Hz), 144.3 (d, J_(C-F)=2.3 Hz), 136.1, 133.3,129.1, 129.0, 117.9 (d, J_(C-F)=3.5 Hz), 53.3, 41.6, 38.5, 28.9, 27.4;HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H2₂Cl₂FN₆S, 443.0988; found443.1007.

6.2.18 Synthesis of Compounds of Formula 106 (Scheme 18)

General Procedure for the Synthesis of 104 a-c.

To 4-amino-1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridine-2-thiol (103)(50 mg, 0.174 mmol) was added respective iodine (0.348 mmol),neocuproine hydrate (3.6 mg, 0.0174 mmol), CuI (3.3 mg, 0.0174 mmol),sodium tert-butoxide (25 mg, 0.261 mmol) and lastly DMF (5 mL) and thereaction mixture was stirred at 110° C. for 24 hours. Then, the solventwas removed under reduced pressure and the crude product was purified bypreparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 10:1) to afford desired compounds104a-c.

2-((3,5-dichlorophenyl)thio)-1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridin-4-amine(104a)

Obtained as pale yellow solid 39% yield. LCMS found m/z 430.97 [M+H]⁺.

1-(4-methoxybenzyl)-2-(naphthalen-1-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(104b)

Obtained as white solid in 38% yield. ¹H NMR (500 MHz, CDCl₃): δ 8.33(d, J=8.1 Hz, 1H), 7.83 (m, 1H), 7.78 (d, J=5.8 Hz, 1H), 7.75 (d, J=8.2Hz, 1H), 7.51-7.55 (m, 2H), 7.44 (d, J=7.2 Hz, 1H), 7.33 (m, 1H), 6.88(d, J=8.6 Hz, 2H), 6.68 (d, J=8.6 Hz, 2H), 6.55 (d, J=5.9 Hz, 1H), 5.25(s, 2H), 5.23 (br s, 2H), 3.73 (s, 3H). LCMS found m/z 413.08 [M+H]⁺.

2-((2,4-dichlorophenyl)thio)-1-(4-methoxybenzyl)-1H-imidazo[4,5-c]pyridin-4-amine(104c)

Obtained as pale yellow solid in 40% yield. ¹H NMR (500 MHz, CDCl₃): δ7.78 (d, J=5.9 Hz, 1H), 7.36 (d, J=2.1 Hz, 1H), 7.02 (dd, J=8.5, 2.1 Hz,1H), 6.98 (d, J=8.6 Hz, 2H), 6.91 (d, J=8.6 Hz, 1H), 6.73 (d, J=8.6 Hz,2H), 6.65 (d, J=6.0 Hz, 1H), 5.30 (s, 2H), 3.75 (s, 3H). LCMS found m/z430.86 [M+H]⁺.

Compound (103) may be prepared as described in U.S. Pat. No. 8,017,780and International Patent Publication No. WO2008115262.

General Procedure for the Synthesis of 105 a-c.

To coupling products (104a-c) (0.067 mmol) were added trifluoroaceticacid (3 mL) and the reaction mixture was stirred at 80° C. for 3 hours.Then, the solvent was removed under reduced pressure and the crudeproduct was purified by preparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 15:1) toafford deprotected compounds 105a-c.

2-((3,5-dichlorophenyl)thio)-1H-imidazo[4,5-c]pyridin-4-amine (105a)

Obtained as yellow solid in 71% yield. ¹H NMR (500 MHz, CD₃OD): δ 7.47(d, J=6.7 Hz, 1H), 7.41-7.43 (m, 3H), 6.96 (d, J=6.7 Hz, 1H). LCMS foundm/z 310.84 [M+H]⁺.

2-(naphthalen-1-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine (105b)

Obtained as yellow solid in 66% yield. LCMS found m/z 292.95 [M+H]⁺.

2-((2,4-dichlorophenyl)thio)-1H-imidazo[4,5-c]pyridin-4-amine (105c)

Obtained as yellow solid in 92% yield. ¹H NMR (500 MHz, CDCl₃:CD₃OD1:1): δ 7.46-7.48 (m, 2H), 7.30 (d, J=8.5 Hz, 1H), 7.02 (dd, J=8.5, 2.2Hz, 1H), 6.85 (d, J=6.4 Hz, 1H). MS m/z 310.8 (M+H)⁺.

2-((3,5-dichlorophenyl)thio)-1-(3-(isopropylamino)propyl)-1H-imidazo[4,5-c]pyridin-4-amine(106a)

To 2-((3,5-dichlorophenyl)thio)-1H-imidazo[4,5-c]pyridin-4-amine (105a)(14.9 mg, 0.0477 mmol) in dry DMF (1.5 mL) was added Cs₂CO₃ (18.6 mg,0.0572 mmol) and lastly 1,3-dibromopropane (24 μL, 0.0238 mmol) and thereaction mixture was stirred at rt for 2 h. The solvent was removedunder reduced pressure and the crude product was purified by preparatoryTLC (CH₂Cl₂:MeOH—NH₃ (7N), 20:1) to afford 7.6 mg (37%) of1-(3-bromopropyl)-2-((3,5-dichlorophenyl)thio)-1H-imidazo[4,5-c]pyridin-4-amine.LCMS found m/z 433.01 [M+H]⁺. To1-(3-bromopropyl)-2-((3,5-dichlorophenyl)thio)-1H-imidazo[4,5-c]pyridin-4-amine(7.6 mg, 0.0175 mmol) in dry DMF was added isopropylamine (30 μL, 0.35mmol) and the reaction mixture was stirred at rt for 24 hours. Then, thesolvent was removed under reduced pressure and the crude product waspurified by preparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 15:1) to afford 5.7mg (79%) of SO-III-154A (106). ¹H NMR (500 MHz, CDCl₃): δ 7.88 (d, J=5.9Hz, 1H), 7.21-7.25 (m, 3H), 6.75 (d, J=5.9 Hz, 1H), 5.26 (br s, 2H),4.26 (t, J=7.1 Hz, 2H), 2.71 (m, 1H), 2.56 (t, J=6.8 Hz, 2H), 1.88 (m,2H), 1.02 (d, J=6.2 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃): δ 151.3, 142.7,141.7, 140.8, 135.9, 135.7, 127.8, 127.5, 127.0, 97.6, 48.8, 44.0, 43.3,30.5, 22.8. HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₈H₂₂N₅SCl₂, 410.0973;found 410.0978.

1-(3-(isopropylamino)propyl)-2-(naphthalen-1-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(106b)

To 2-(naphthalen-1-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine (105b) (12.6mg, 0.043 mmol) in dry DMF (1.5 mL) was added Cs₂CO₃ (16.8 mg, 0.0517mmol) and lastly 1,3-dibromopropane (17.4 μL, 0.172 mmol) and thereaction mixture was stirred at rt for 1 hour and a half. The solventwas removed under reduced pressure and the crude product was purified bypreparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 15:1) to afford 9.8 mg (55%) of1-(3-bromopropyl)-2-(naphthalen-1-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine.LCMS found m/z 412.95 [M+H]⁺. To1-(3-bromopropyl)-2-(naphthalen-1-ylthio)-1H-imidazo[4,5-c]pyridin-4-amine(9.8 mg, 0.0237 mmol) in dry DMF was added isopropylamine (50.9 μL,0.592 mmol) and the reaction mixture was stirred at rt for 24 hours.Then, the solvent was removed under reduced pressure and the crudeproduct was purified by preparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 15:1) toafford 5.6 mg (60%) of SO-IV-03A (106b). ¹H NMR (600 MHz, CDCl₃): δ 8.40(d, J=8.4 Hz, 1H), 7.88 (d, J=8.1 Hz, 1H), 7.82 (m, 2H), 7.55-7.62 (m,2H), 7.48 (d, J=7.3 Hz, 1H), 7.38 (t, J=7.7 Hz, 1H), 6.69 (d, J=5.9 Hz,1H), 5.21 (br s, 2H), 4.18 (t, J=7.3 Hz, 2H), 2.62 (m, 1H), 2.42 (t,J=6.9 Hz, 2H), 1.71-1.75 (m, 2H), 0.96 (d, J=6.2 Hz, 6H). ¹³C NMR (150MHz, CDCl₃): δ 151.0, 145.3, 141.2, 141.0, 134.1, 132.4, 129.9, 129.1,128.9, 128.7, 127.3, 127.2, 126.7, 125.9, 124.5, 97.6, 48.7, 44.0, 43.3,30.3, 22.9. LCMS found m/z 392.13 [M+H]⁺.

2-((2,4-dichlorophenyl)thio)-1-(3-(isopropylamino)propyl)-1H-imidazo[4,5-c]pyridin-4-amine(106c)

To 2-((2,4-dichlorophenyl)thio)-1H-imidazo[4,5-c]pyridin-4-amine (105c)(20 mg, 0.064 mmol) in dry DMF (2 mL) was added Cs₂CO₃ (25.2 mg, 0.0768mmol) and lastly 1,3-dibromopropane (32.7 μL, 0.323 mmol) and thereaction mixture was stirred at rt for 2 h. Then another portion ofCs₂CO₃ (40 mg, 0.122 mmol) and 1,3-dibromopropane (20 μL) were added andthe reaction mixture was stirred for 1 more hour. The solvent wasremoved under reduced pressure and the crude product was purified bypreparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 20:1, 2×) to afford 11.6 mg (42%)of1-(3-bromopropyl)-2-((2,4-dichlorophenyl)thio)-1H-imidazo[4,5-c]pyridin-4-amine.LCMS found m/z 432.81 [M+H]⁺. To1-(3-bromopropyl)-2-((2,4-dichlorophenyl)thio)-1H-imidazo[4,5-c]pyridin-4-amine(11.6 mg, 0.0268 mmol) in dry DMF was added isopropylamine (110 μL, 1.34mmol) and the reaction mixture was stirred at rt for 24 hours. Then, thesolvent was removed under reduced pressure and the crude product waspurified by preparatory TLC (CH₂Cl₂:MeOH—NH₃ (7N), 20:1) to afford 8.3mg (75%) of HJP-VI-101 (106c). ¹H NMR (500 MHz, CDCl₃): δ 7.86 (d, J=5.9Hz, 1H), 7.43 (d, J=2.2 Hz, 1H), 7.13 (dd, J=8.5, 2.2 Hz, 1H), 7.02 (d,J=8.6 Hz, 1H), 6.75 (d, J=5.9 Hz, 1H), 5.29 (br s, 2H), 4.25 (t, J=7.1Hz, 2H), 2.71 (m, 1H), 2.56 (t, J=6.8 Hz, 2H), 1.88 (m, 2H), 1.02 (d,J=6.3 Hz, 6H). LCMS found m/z 410.08 [M+H]⁺.

6.2.19 Synthesis of Compounds of Formula 110-111 (Scheme 19)

General Procedure for the Synthesis of 108 a and 108 b.

8-Mercaptoadenine (3.6 mmol), neocuproine hydrate (0.36 mmol), CuI (0.36mmol), NaO-t-Bu (7.2 mmol), respective aryliodide (10.8 mmol), andanhydrous DMF (24 mL) were taken in a round bottom flask flushed withnitrogen. The flask was sealed with Teflon tape, heated at 110° C., andmagnetically stirred for 24 h under nitrogen. Solvent was removed underreduced pressure and the resulting residue was chromatographed(CH₂Cl₂:MeOH:AcOH, 20:1:0.5).

8-((4-chloro-2-fluorophenyl)thio)-9H-purin-6-amine (108 a)

Obtained as a light yellow solid in 49% yield. MS (ESI): m/z 296.1[M+H]⁺.

8-((2-chloro-4-fluorophenyl)thio)-9H-purin-6-amine (108 b)

Obtained as a light yellow solid in 49% yield. MS (ESI): m/z 296.1[M+H]⁺.

General Procedure for Synthesis of N9 Alkylated 8-aryl SulfanylDerivatives 109a and 109b.

8-Arylsulfanyl adenine (108a or 108b, 1.21 mmol) was dissolved in DMF(15 mL) and Cs₂CO₃ (1.45 mmol) and 1,3-dibromopropane (2.42 mmol) wereadded and the mixture was stirred under nitrogen at for 2-4 h. Solventwas removed under reduced pressure and the resulting residue waschromatographed (CH₂Cl₂:MeOH:AcOH, 20:1:0.5) to afford desired compounds109 a and 109b.

9-(3-bromopropyl)-8-((4-chloro-2-fluorophenyl)thio)-9H-purin-6-amine(109a)

Obtained as a solid in 35% yield. MS (ESI): m/z 415.9 [M+H]⁺.

9-(3-bromopropyl)-8-((2-chloro-4-fluorophenyl)thio)-9H-purin-6-amine(109b)

Obtained as a solid in 29% yield. MS (ESI): m/z 415.9 [M+H]⁺.

General Procedure for the Synthesis of 110 and 111

A mixture of 109a or 109b (12 mg, 0.028 mmol) and amine (1.40 mmol, 50equiv.) in DMF (1 mL) under nitrogen protection was stirred at roomtemperature for 16-24 hrs. Following solvent removal, the crude materialwas purified by preparative TLC (CH₂Cl₂:CH₃OH—NH₃ (7N), 20:1 or 15:1) toafford desired products 110-111.

8-((4-chloro-2-fluorophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(110, HJP-VI-23)

Yield 15%. ¹H NMR (600 MHz, CDCl₃+5 drops CD₃OD) δ 8.23 (s, 1H), 7.62(t, J=7.9 Hz, 1H), 7.27-7.32 (m, 2H), 4.42 (t, J=6.7 Hz, 2H), 3.26-3.29(m, 1H), 3.00 (t, J=7.4 Hz, 2H), 2.31-2.37 (m, 2H), 1.35 (d, J=6.5 Hz,6H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) δ 162.9, 161.2, 150.6,150.5, 149.5, 145.6, 138.8 (d, J-F=9.8 Hz), 137.3, 126.1 (d, J-F=3.5Hz), 117.8 (d, J-F=25.5 Hz), 112.4 (d, J_(C-F)=18.3 Hz), 50.8, 41.8,41.1, 26.1, 19.0; HRMS (ESI) m/z [M+H]⁺ calcd. for C₁₇H₂₁ClFN₆S,395.1221; found 395.1216.

8-((2-chloro-4-fluorophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(111, HJP-VI-25)

Yield 16%. ¹H NMR (600 MHz, CDCl₃) δ 8.31 (s, 1H), 7.34 (dd, J=8.8 and5.8 Hz, 1H), 7.23 (dd, J=8.2 and 2.7 Hz, 1H), 6.95 (dt, J=7.9 and 2.7Hz, 1H), 6.24 (br s, 2H), 4.32 (t, J=7.0 Hz, 2H), 2.68-2.72 (m, 1H),2.56 (t, J=6.9 Hz, 2H), 1.95-1.99 (m, 2H), 1.02 (d, J=6.2 Hz, 6H); ¹³CNMR (150 MHz, CDCl₃) δ 163.3, 161.6, 154.7, 153.0, 151.6, 144.7, 136.4(d, J_(C-F)=10.4 Hz), 134.2 (d, J_(C-F)=8.8 Hz), 125.7 (d, J_(C-F)=3.8Hz), 120.1, 118.1 (d, J_(C-F)=25.1 Hz), 115.3 (d, J_(C-F)=21.7 Hz),48.7, 43.7, 41.7, 30.3, 22.9; HRMS (ESI) m/z [M+H]⁺ calcd. forC₁₇H₂₁ClFN₆S, 395.1221; found 395.1222.

6.3 Synthesis of PU-H71-Type Fluorescently Labeled Probes

2-(4-(6-Amino-8-((6-iodobenzo[d][1,3]dioxol-5-yl)thio)-9H-purin-9-yl)butyl)isoindoline-1,3-dione(113b

200 mg (0.484 mmol) of 112 was dissolved in DMF (8 mL). 466 mg (1.43mmol) of Cs₂CO₃ and 683 mg (2.42 mmol) of N-(4-bromobutyl)phthalimidewere added and the mixture was sonicated for 30 min. 31.5 mg (0.097mmol) of Cs₂CO₃ was added and the mixture was again sonicated for 30min. This was repeated two more times for a total reaction time of 2 h.DMF was removed and the resulting residue was purified by preparatoryTLC (CH₂Cl₂:MeOH:AcOH, 15:1:0.5) to give 134 mg (45%) of 113b. ¹H NMR(500 MHz, CDCl₃) δ 8.18 (S, 1H), 7.84 (dd, J=5.5, 3.1 Hz, 2H), 7.72 (dd,J=5.5, 3.1 Hz, 2H), 7.22 (s, 1H), 6.89 (s, 1H), 6.76 (br s, 2H), 5.99(s, 2H), 4.23 (t, J=7.1 Hz, 2H), 3.69 (t, J=7.0 Hz, 2H), 1.67-1.83 (m,4H); MS (ESI) m/z 615.2 [M+H]⁺.

9-(4-Aminobutyl)-8-((6-iodobenzo[d][1,3]dioxol-5-yl)thio)-9H-purin-6-amine(114b)

To a suspension of 113b (38.9 mg, 0.063 mmol) in 2 mL MeOH/CH₂Cl₂ (7:1mL) was added hydrazine hydrate (46 μL, 0.950 mmol) and the mixture wasstirred at rt for 12 h. Solvent was removed under reduced pressure andthe resulting residue was purified by preparatory TLC (CH₂Cl₂:MeOH—NH₃(7N), 10:1) to give 18 mg (59%) of 114b. ¹H NMR (500 MHz, CDCl₃/MeOH-d₄)δ 8.22 (s, 1H), 7.38 (s, 1H), 7.04 (s, 1H), 6.05 (s, 2H), 4.23 (t, J=7.4Hz, 2H), 2.78 (t, J=7.1 Hz, 2H), 1.82-1.91 (m, 2H), 1.55-1.63 (m, 2H);MS (ESI) m/z 485.0 [M+H]⁺.

PU-C4-FITC (115b)

114b (9.7 mg, 0.020 mmol), FITC (8.57 mg (0.022 mmol) and Et₃N (0.1 mL)in DMF (0.2 mL) was stirred for 3 h at rt. The reaction mixture wasdirectly purified by HPLC to give 5.2 mg (30%) of 115b. ¹H NMR (600 MHz,MeOH-d₄) δ 8.22 (s, 1H), 8.00 (s, 1H), 7.61 (d, J=7.6 Hz, 1H), 7.37 (s,1H), 7.19 (s, 1H), 7.06 (d, J=8.2 Hz, 1H), 6.58-6.67 (m, 4H), 6.48 (dd,J=8.7, 2.0 Hz, 2H), 5.97 (s, 2H), 4.30 (t, J=7.0 Hz, 2H), 3.58 (br s,2H), 1.90-2.00 (m, 2H), 1.61-1.70 (m, 2H); HRMS (ESI) m/z [M+H]⁺ calcd.for C₃₇H₂₉IN₇O₇S₂, 874.0615; found 874.0610; HPLC Rt=9.57 (98%).

2-(6-(6-Amino-8-((6-iodobenzo[d][1,3]dioxol-5-yl)thio)-9H-purin-9-yl)hexyl)isoindoline-1,3-dione(113c)

200 mg (0.484 mmol) of 112 was dissolved in DMF (8 mL). 466 mg (1.43mmol) of Cs₂CO₃ and 751 mg (2.42 mmol)N-(6-bromohexyl)phthalimide wereadded and the mixture was sonicated for 2 h. Solvent was removed underreduced pressure and the resulting residue was purified by preparatoryTLC (CH₂Cl₂:MeOH:AcOH, 15:1:0.5) to give 100 mg (32%) of 113c. ¹H NMR(500 MHz, CDCl₃) δ 8.26 (s, 1H), 7.83 (dd, J=5.4, 3.1 Hz, 2H), 7.70 (dd,J=5.4, 3.0 Hz, 2H), 7.26 (s, 1H), 6.87 (s, 1H), 6.36 (br s, 2H), 5.96(s, 2H), 4.18 (t, J=7.5 Hz, 2H), 3.66 (t, J=7.2 Hz, 2H), 1.70-1.79 (m,2H), 1.60-1.68 (m, 2H), 1.32-1.43 (m, 4H); MS (ESI) m/z 643.2 [M+H]⁺.

9-(6-Aminohexyl)-8-((6-iodobenzo[d][1,3]dioxol-5-yl)thio)-9H-purin-6-amine(114c)

To a suspension of 113c (97 mg, 0.1511 mmol) in 4 mL MeOH/CH₂Cl₂ (7:1mL) was added hydrazine hydrate (110 μL, 2.27 mmol) and the mixture wasstirred at rt for 12 h. Solvent was removed under reduced pressure andthe resulting residue was purified by preparatory TLC (CH₂Cl₂:MeOH—NH₃(7N), 10:1) to give 47 mg (61%) of 114c. ¹H NMR (500 MHz, CDCl₃) δ 8.32(s, 1H), 7.31 (s, 1H), 6.90 (s, 1H), 5.99 (s, 2H), 5.84 (br s, 2H), 4.20(t, J=7.5 Hz, 2H), 2.67 (t, J=6.5 Hz, 2H), 1.72-1.84 (m, 2H), 1.31-1.45(m, 6H); MS (ESI) m/z 513.0 [M+H]⁺.

PU-C6-FITC (115c)

114c (9.7 mg, 0.01894 mmol), FITC (8.11 mg, 0.0208 mmol) and Et₃N (0.1mL) in DMF (0.2 mL) was stirred for 3 h at rt. The reaction mixture wasdirectly purified by HPLC to give 8.0 mg (47%) of 115c. ¹H NMR (600 MHz,MeOH-d₄) δ 8.23 (s, 1H), 8.09 (s, 1H), 7.65 (d, J=7.9 Hz, 1H), 7.35 (s,1H), 7.16 (s, 1H), 7.08 (d, J=8.3 Hz, 1H), 6.71 (d, J=8.8 Hz, 2H), 6.67(d, J=2.2 Hz, 2H), 6.53 (dd, J=8.8, 2.2 Hz, 2H), 5.96 (s, 2H), 4.24 (t,J=7.1 Hz, 2H), 3.50 (br s, 2H), 1.79-1.88 (m, 2H), 1.52-1.61 (m, 2H),1.31-1.42 (m, 4H); HRMS (ESI) m/z [M+H]⁺ calcd. for C39H₃₃IN₇O₇S₂,902.0928; found 902.0939; HPLC Rt=10.02 (99%).

2-(8-(6-Amino-8-((6-iodobenzo[d][1,3]dioxol-5-yl)thio)-9H-purin-9-yl)octyl)isoindoline-1,3-dione(113d)

200 mg (0.484 mmol) of 112 was dissolved in DMF (8 mL). 466 mg (1.43mmol) of Cs₂CO₃ and 819 mg (2.42 mmol)N-(8-bromooctyl)phthalimide wereadded and the mixture was sonicated for 1.5 h. Solvent was removed underreduced pressure and the resulting residue was purified by preparatoryTLC (CH₂Cl₂:MeOH:AcOH, 15:1:0.5) to give 120 mg (34%) of 113d. ¹H NMR(500 MHz, CDCl₃) δ 8.29 (s, 1H), 7.84 (dd, J=5.5, 3.1 Hz, 2H), 7.70 (dd,J=5.5, 3.1 Hz, 2H), 7.28 (s, 1H), 6.87 (s, 1H), 6.29 (br s, 2H), 5.96(s, 2H), 4.18 (t, J=7.5 Hz, 2H), 3.67 (t, J=7.3 Hz, 2H), 1.62-1.77 (m,4H), 1.25-1.36 (m, 8H); MS (ESI) m/z 671.3 [M+H]⁺.

9-(8-Aminooctyl)-8-((6-iodobenzo[d][1,3]dioxol-5-yl)thio)-9H-purin-6-amine(114d)

To a suspension of 113d (90.1 mg, 0.1345 mmol) in 4 mL MeOH/CH₂Cl₂ (7:1mL) was added hydrazine hydrate (98 μL, 2.017 mmol) and the mixture wasstirred at rt for 12 h. Solvent was removed under reduced pressure andthe resulting residue was purified by preparatory TLC (CH₂Cl₂:MeOH—NH₃(7N), 10:1) to give 25 mg (34%) of 114d. ¹H NMR (500 MHz, CDCl₃) δ 8.33(s, 1H), 7.31 (s, 1H), 6.90 (s, 1H), 5.99 (s, 2H), 5.72 (br s, 2H), 4.20(t, J=7.5 Hz, 2H), 2.66 (t, J=7.1 Hz, 2H), 1.70-1.80 (m, 2H), 1.36-1.45(m, 2H), 1.21-1.35 (m, 8H); MS (ESI) m/z 541.1 [M+H]⁺.

PU-C8-FITC (115d)

114d (15.0 mg, 0.028 mmol), FITC (11.9 mg, 0.031 mmol) and Et₃N (0.1 mL)in DMF (0.2 mL) was stirred for 4 h at rt. The reaction mixture wasdirectly purified by HPLC to give 16.9 mg (66%) of 115d. ¹H NMR (600MHz, MeOH-d₄) δ 8.22 (s, 1H), 8.11 (s, 1H), 7.68 (d, J=7.8 Hz, 1H), 7.34(s, 1H), 7.12 (s, 1H), 7.09 (d, J=8.2 Hz, 1H), 6.72 (d, J=8.7 Hz, 2H),6.67 (d, J=2.0 Hz, 2H), 6.53 (dd, J=8.7, 2.0 Hz, 2H), 5.96 (s, 2H), 4.20(t, J=7.1 Hz, 2H), 3.50 (br s, 2H), 1.74-1.81 (m, 2H), 1.52-1.59 (m,2H), 1.23-1.35 (m, 8H); HRMS (ESI) m/z [M+H]⁺ calcd. for C₄₁H₃₇IN₇O₇S₂,930.1241; found 930.1231; HPLC Rt=10.60 (96%).

6.4. Synthesis of PU-WS13 Beads

9-(3-Bromopropyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (116)

To a solution of 15 (0.4 g, 1.29 mmol) in 20 ml of dry DMF was added0.65 g (2.00 mmol, 1.55 equiv.) of Cs₂CO₃ and allowed to stir at roomtemperature for 15 minutes. Then 0.9 g (4.47 mmol, 3.5 equiv.) of1,3-dibromopropane was added and the reaction mixture was stirred for 2hrs at room temperature. Solvent was removed under reduced pressure andthe residue purified by column chromatography (CH₂Cl₂:CH₃OH:CH₃COOH;20:1:0.1) to yield 0.15 g (27%) of desired N-9 isomer (116). ¹H NMR (500MHz, CDCl₃) δ 8.36 (s, 1H), 7.31 (m, 3H), 4.31 (t, J=7.1 Hz, 2H), 3.15(t, J=6.7 Hz, 2H), 2.32 (quintet, J=6.8 Hz, 2H); ¹³C NMR (125 MHz,CDCl₃) 154.7, 153.6, 151.6, 143.7, 135.9, 134.3, 128.6, 128.3, 120.3,42.6, 33.0, 29.3. MS (ESI) m/z 432.1 [M+H]⁺.

Tert-Butyl-(6-((3-(6-amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)amino)hexyl)carbamate(117)

Compound 116 (0.15 g, 0.348 mmol) and tert-Butyl 6-aminohexylcarbamate(0.752 g, 3.48 mmol) in DMF (5 mL) was stirred at rt for 24 h. Thereaction mixture was concentrated and the residue purified bypreparative TLC [CH₂Cl₂/MeOH—NH₃ (7 N), 20:1] to give 81 mg (41%) of 117as a yellow solid. ¹H NMR (400 MHz, CDCl₃/MeOH-d₄, 6) 8.19 (s, 1H), 7.46(d, J=1.7 Hz, 2H), 7.44 (d, J=1.6 Hz, 1H), 4.31 (t, J=6.9 Hz, 2H), 3.05(m, 2H), 2.56 (t, J=6.7 Hz, 2H), 2.51 (t, J=7.0 Hz, 2H), 1.99 (m, 2H),1.44 (m, 13H), 1.30 (m, 4H); MS (ESI): m/z 568.2 [M+H]⁺.

N1-(3-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)hexane-1,6-diamine(118)

Compound 117 (81 mg, 0.143 mmol) was dissolved in 10 mL of CH₂Cl₂/TFA(4:1) and the solution was stirred at room temperature for 45 min.Solvent was removed under reduced pressure and the residue purified bypreparative TLC [CH₂Cl₂/MeOH—NH₃ (7N), 20:1-10:1] to give 41 mg (62%yield) of 118 as a white solid. ¹H NMR (400 MHz, CDCl₃, δ) 8.34 (s, 1H),7.32 (m, 3H), 6.04 (bs, 2H), 4.31 (t, J=7.0 Hz, 2H), 2.49-2.51 (m, 4H),1.94-2.03 (m, 2H), 1.31-1.44 (m, 12H); MS (ESI): m/z 468.3 [M+H]⁺.

Compound-Affi-Gel® 10 Beads (119)

118 (41 mg, 0.087 mmol) was dissolved in DMF (4 mL) and added to 10 mLof Affi-Gel® 10 beads (prewashed, 3×20 mL DMF) in a solid phase peptidesynthesis vessel. 75 μL of N,N-diisopropylethylamine and severalcrystals of DMAP were added and this was shaken at room temperature for2.5 h. Then 2-methoxyethylamine (17.5 mg, 20 μl, 0.23 mmol) was addedand shaking was continued for 30 min. Then the solvent was removed andthe beads washed for 10 min each time with CH₂Cl₂/Et₃N (9:1, 4×20 mL),DMF (3×20 mL), Felts buffer (3×20 mL) and i-PrOH (3×20 mL). The beads119 were stored in i-PrOH (beads/i-PrOH (1:2), v/v) at −80° C.

6.5 Synthesis of PU-WS13 Biotin Analogs and Fluorescently Labeled Probes

2-(3-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)isoindoline-e-1,3-dione(120a)

To a solution of 15 (0.4 g, 1.29 mmol) in 20 ml of dry DMF was added0.65 g (2.00 mmol, 1.55 equiv.) of Cs₂CO₃ and allowed to stir at roomtemperature for 15 minutes. Then 1.2 g (4.47 mmol, 3.5 equiv.) ofbromopropyl phthalamide was added and the reaction mixture was stirredfor 2 hrs at room temperature. Solvent was removed under reducedpressure and the residue purified by column chromatography(CH₂C12:CH₃OH:CH₃COOH; 20:1:0.1) to yield 0.15 g (25%) of desired N-9isomer (120a). ¹H NMR (500 MHz, CDCl₃) δ 8.16 (s, 1H), 7.82-7.86 (m,2H), 7.71-7.75 (m, 2H), 7.24 (t, J=1.65 Hz, 1H), 7.18 (t, J=1.6 Hz, 1H),4.31 (t, J=7.5 Hz, 2H), 3.37 (t, J=6.7 Hz, 2H), 2.21 (quintet, J=6.8 Hz,2H); ¹³C NMR (125 MHz, CDCl₃) 176.1, 168.1, 155.1, 152.2, 150.8, 143.6,135.8, 134.1, 133.9, 131.8, 128.6, 128.2, 123.3, 41.8, 35.3, 28.6. MS(ESI) m/z 498.95/501.13 [M+H]⁺.

9-(3-Aminopropyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (121a)

To a solution of 120a (0.15 g, 0.3 mmol) in 14 ml CH₂Cl₂+2 ml CH₃OH wasadded 194 μL (4.03 mmol, 15 equiv.) of hydrazine hydrate and allowed tostir at room temperature for 12 h. Solvent was removed under reducedpressure and the residue purified by column chromatography (CH₂Cl₂:CH₃OH—NH₃ (7N); 20:1) to yield 65 mg (66%) of 121a. ¹H NMR (500 MHz,CDCl₃+5 drops CD₃OD) δ 8.26 (s, 1H), 7.34-7.39 (m, 3H), 4.31 (t, J=6.9Hz, 2H), 2.65 (t, J=6.6 Hz, 2H), 1.93 (quintet, J=6.8 Hz, 2H); ¹³C NMR(125 MHz, CDCl₃) 154.6, 152.9, 151.2, 144.3, 135.9, 133.2, 129.1, 128.9,119.6, 40.9, 37.9, 32.6. MS (ESI) m/z 369.14/371.22 [M+H]⁺.

N-(3-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)-1-(5-((3aS,4R,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-3,6,9,12-tetraoxapentadecan-15-amide(122a)

121a (18 mg, 0.0487 mmol), EZ-Link® NHS-PEG₄-Biotin (31.6 mg, 0.0536mmol) and DIEA (12.6 mg, 16.9 μL 0.0974 mmol) in DMF (1.5 ml) wasstirred at rt for 1 h. The reaction mixture was concentrated underreduced pressure and resulting residue was purified by preparatory TLC(CH₂Cl₂-MeOH—NH₃ (7N), 10:1) to give 30 mg (73%) of 122a. ¹H NMR (600MHz, CDCl₃) δ 8.30 (s, 1H), 7.51 (t, J=5.8 Hz, 1H), 7.26-7.29 (m, 3H),7.05 (t, J=4.9 Hz, 1H), 6.82 (s, 1H), 6.63 (s, 1H), 6.00 (s, 1H),4.79-4.50 (m, 1H), 4.29-4.32 (m, 1H), 4.28 (t, J=6.7 Hz, 2H), 3.77 (t,J=6.1 Hz, 2H), 3.59-3.65 (m, 12H), 3.55 (t, J=5.0 Hz, 2H), 3.40-3.43 (m,2H), 3.18 (q, J=6.1 Hz, 2H), 3.11-3.15 (m, 1H), 2.86-2.90 (m, 2H), 2.52(t, J=6.0 Hz, 2H), 2.19 (t, J=7.4 Hz, 2H), 1.94 (quintet, J=6.7 Hz, 2H),1.69-1.76 (m, 2H), 1.58-1.67 (m, 2H), 1.38-1.44 (m, 2H); HRMS (ESI) m/z[M+H]⁺ calcd. for C₃₅H₅₀Cl₂N₉O₇S₂, 842.2652; found 842.2657; HPLC(Method A) Rt=8.29.

N-(3-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)-6-(6-(5-((3aS,4R,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide(123a)

121a (5 mg, 0.0128 mmol), EZ-Link® NHS-LC_LC-Biotin (10.2 mg, 0.018mmol) and DIEA (4.21 mg, 5.7 μL, 0.0326 mmol) in DMF (0.5 ml) wasstirred at rt for 1 h. The reaction mixture was concentrated underreduced pressure and resulting residue was purified by preparatory TLC(CH₂Cl₂-MeOH—NH₃ (7N), 10:1) to give 6.3 mg (60%) of desired compound.¹H NMR (500 MHz, CDCl₃+3 drops of CD₃OD) δ 8.26 (s, 1H), 7.38 (t, J=1.7Hz, 1H), 7.35 (d, J=1.8 Hz, 2H), 4.48-4.52 (m, 1H), 4.29-4.33 (m, 1H),4.26 (t, J=6.9 Hz, 2H), 3.09-3.15 (m, 8H), 2.24 (t, J=6.9 Hz, 2H),2.10-2.20 (m, 8H), 1.94 (quintet, J=6.3 Hz, 2H), 1.58-1.71 (m, 10H),1.45-1.53 (m, 4H), 1.41-1.44 (m, 3H); HRMS (ESI) m/z [M+H]⁺ calcd. forC₃₆H₅₁Cl₂N₁₀O₄S₂, 821.2913; found 821.2941; HPLC (Method A) R_(t)=9.92.

2-(6-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)hexyl)isoindoline-1,3-dione(120b)

To a solution of 15 (0.4 g, 1.29 mmol) in 20 ml of dry DMF was added0.75 g (2.31 mmol, 1.8 equiv.) of Cs₂CO₃ and allowed to stir at roomtemperature for 15 minutes. Then 1.4 g (4.5 mmol, 3.5 equiv.) ofbromohexyl phthalamide was added and the reaction mixture was stirredfor 4 hrs at room temperature. Solvent was removed under reducedpressure and the residue purified by column chromatography(CH₂Cl₂:CH₃OH:CH₃COOH; 20:1:0.1) to yield 0.10 g (15%) of desired N-9isomer (120b). ¹H NMR (500 MHz, CDCl₃) δ 8.25 (s, 1H), 7.83-7.85 (m,2H), 7.73-7.74 (m, 2H), 7.32 (m, 3H), 4.21 (t, J=7.4 Hz, 2H), 3.66 (t,J=7.2 Hz, 2H), 1.75-1.78 (m, 2H), 1.62-1.65 (m, 2H), 1.32-1.38 (m, 4H).MS (ESI) m/z 541.33/543.24 [M+H]⁺.

9-(3-Aminohexyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (121b)

To a solution of 120b (0.10 g, 0.18 mmol) in 10 ml CH₂Cl₂+1.5 ml CH₃OHwas added 140 μL (2.77 mmol, 15 equiv.) of hydrazine hydrate and allowedto stir at room temperature for 12 h. Solvent was removed under reducedpressure and the residue purified by column chromatography (CH₂Cl₂:CH₃OH—NH₃ (7N); 20:1) to yield 56 mg (74%) of 121b. ¹H NMR (600 MHz,CDCl₃+5 drops CD₃OD) δ 8.31 (s, 1H), 7.31-7.34 (m, 3H), 4.22 (t, J=7.9Hz, 2H), 2.65 (t, J=7.3 Hz, 2H), 1.76-1.78 (m, 2H), 1.41-1.43 (m, 2H),1.31-1.36 (m, 4H); ¹³C NMR (150 MHz, CDCl₃+5 drops CD₃OD) 154.8, 153.2,151.2, 143.8, 135.9, 134.1, 128.7, 128.6, 119.9, 43.8, 41.6, 33.0, 29.7,26.4, 26.3. MS (ESI) m/z 411.24/413.24 [M+H]⁺.

N-(6-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)hexyl)-1-(5-((3aS,4R,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-3,6,9,12-tetraoxapentadecan-15-amide(122b): 121b

(6.4 mg, 0.0156 mmol), EZ-Link® NHS-PEG₄-Biotin (10.1 mg, 0.017 mmol)and DIEA (4 mg, 5.5 μL, 0.031 mmol) in DMF (0.5 ml) was stirred at rtfor 1 h. The reaction mixture was concentrated under reduced pressureand resulting residue was purified by preparatory TLC (CH₂Cl₂-MeOH—NH₃(7N), 10:1) to give 9.6 mg (77%) of 122b. ¹H NMR (600 MHz, CD₂Cl₂) δ8.27 (s, 1H), 7.30 (t, J=1.8 Hz, 1H), 7.28 (d, J=1.8 Hz, 2H), 6.76 (t,J=5.2 Hz, 1H), 6.59 (t, J=5.3 Hz, 1H), 6.46 (s, 1H), 6.35 (s, 2H), 5.54(s, 1H), 4.45-4.49 (m, 1H), 4.27-4.31 (m, 1H), 4.20 (t, J=7.3 Hz, 2H),3.68 (t, J=5.9 Hz, 2H), 3.55-3.59 (m, 14H), 3.53 (t, J=5.1 Hz, 2H), 3.38(q, J=5.1 Hz, 2H), 3.14 (q, J=7.1 Hz, 3H), 2.87-2.91 (m, 1H), 2.67-2.73(m, 1H), 2.39 (t, J=6.0 Hz, 2H), 2.16 (t, J=7.4 Hz, 2H), 1.57-1.76 (m,7H), 1.37-1.44 (m, 5H); HRMS (ESI) m/z [M+H]⁺ calcd. forC₃₈H₅₆Cl₂N₉O₇S₂, 884.3121; found 884.3157; HPLC (Method A) Rt=9.00.

N-(6-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)hexyl)-6-(6-(5-((3aS,4R,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide(123b)

121b (5 mg, 0.0122 mmol), EZ-Link® NHS-LC_LC-Biotin (9.65 mg, 0.0146mmol) and DIEA (4 mg, 5.5 μL, 0.031 mmol) in DMF (0.5 ml) was stirred atrt for 1 h. The reaction mixture was concentrated under reduced pressureand resulting residue was purified by preparatory TLC (CH₂Cl₂-MeOH—NH₃(7N), 10:1) to give 4.2 mg (42%) of 123b. ¹H NMR (600 MHz, CD₂Cl₂+5drops of CD₃OD) δ 8.16 (s, 1H), 7.26-7.31 (m, 3H), 4.38-4.42 (m, 1H),4.20-4.23 (m, 1H), 4.13 (t, J=7.3 Hz, 2H), 3.03-3.09 (m, 8H), 2.80-2.85(m, 1H), 2.61-2.65 (m, 1H), 2.03-2.11 (m, 7H), 1.25-1.69 (m, 24H); HRMS(ESI) m/z [M+H]⁺ calcd. for C₃₉H₅₇Cl₂N₁₀O₄S₂, 863.3383; found 863.3402;HPLC (Method A) Rt=9.47.

2-(6-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)octyl)isoindoline-1,3-dione(120c)

To a solution of 15 (0.4 g, 1.29 mmol) in 20 ml of dry DMF was added0.75 g (2.31 mmol, 1.8 equiv.) of Cs₂CO₃ and allowed to stir at roomtemperature for 15 minutes. Then 1.5 g (4.48 mmol, 3.5 equiv.) ofbromooctyl phthalamide was added and the reaction mixture was stirredfor 2 hrs at room temperature. Solvent was removed under reducedpressure and the residue purified by column chromatography(CH₂Cl₂:CH₃OH:CH₃COOH; 20:1:0.1) to yield 0.15 g (21%) of desired N-9isomer (120c). ¹H NMR (500 MHz, CDCl₃) δ 8.18 (s, 1H), 7.71-7.75 (m,2H), 7.60-7.64 (m, 2H), 7.21 (m, 3H), 4.13 (t, J=7.3 Hz, 2H), 3.57 (t,J=7.2 Hz, 2H), 1.55-1.64 (m, 4H), 1.19-1.21 (m, 8H); ¹³C NMR (125 MHz,CDCl₃) 174.5, 167.6, 153.1, 149.9, 149.6, 144.1, 134.9, 133.0, 132.5,131.0, 128.1, 127.9, 122.2, 48.9, 43.3, 36.9, 28.6, 27.9, 27.5, 25.7,25.5. MS (ESI) m/z 569.22/571.13 [M+H]⁺.

9-(3-Aminohexyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (121c)

To a solution of 120c (0.15 g, 0.26 mmol) in 10 ml CH₂Cl₂+1.5 ml CH₃OHwas added 194 μL (3.90 mmol, 15 equiv.) of hydrazine hydrate and allowedto stir at room temperature for 12 h. Solvent was removed under reducedpressure and the residue purified by column chromatography (CH₂Cl₂:CH₃OH—NH₃ (7N); 20:1) to yield 57 mg (50%) of 121c. ¹H NMR (600 MHz,CDCl₃+5 drops CD₃OD) δ 8.26 (s, 1H), 7.12-7.16 (m, 3H), 4.12 (t, J=7.3Hz, 2H), 2.64 (t, J=6.9 Hz, 2H), 1.62-1.68 (m, 2H), 1.35-1.41 (m, 2H),1.13-1.20 (m, 8H); ¹³C NMR (125 MHz, CDCl₃+5 drops CD₃OD) 155.3, 153.4,151.3, 142.8, 135.7, 134.9, 128.1, 127.8, 120.3, 43.9, 41.5, 30.9, 29.8,29.1, 28.9, 26.7, 26.5. MS (ESI) m/z 439.16/441.15 [M+H]⁺.

N-(8-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)octyl)-1-(5-((3aS,4R,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)-3,6,9,12-tetraoxapentadecan-15-amide(122c)

121c (5.7 mg, 0.013 mmol), EZ-Link® NHS-PEG₄-Biotin (8.4 mg, 0.014 mmol)and DIEA (3.4 mg, 4.5 μL, 0.026 mmol) in DMF (0.5 ml) was stirred at rtfor 1 h. The reaction mixture was concentrated under reduced pressureand resulting residue was purified by preparatory TLC (CH₂Cl₂-MeOH—NH₃(7N), 10:1) to give 6.6 mg (56%) of 122c. ¹H NMR (600 MHz, CD₂Cl₂+5drops of CD₃OD) δ 8.15 (s, 1H), 7.30 (t, J=1.7 Hz, 1H), 7.26 (d, J=1.7Hz, 2H), 4.38-4.43 (m, 1H), 4.20-4.23 (m, 1H), 4.13 (t, J=7.4 Hz, 2H),3.61 (t, J=6.0 Hz, 2H), 3.50-3.55 (m, 14H), 3.45 (t, J=5.3 Hz, 2H),3.27-3.31 (m, 4H), 3.07 (t, J=7.3 Hz, 3H), 2.81-2.85 (m, 1H), 2.60-2.64(m, 1H), 2.34 (t, J=6.0 Hz, 2H), 2.12 (t, J=7.6 Hz, 2H), 1.49-1.67 (m,8H), 1.30-1.39 (m, 6H); HRMS (ESI) m/z [M+H]⁺ calcd. forC₄₀H₆₀Cl₂N₉O₇S₂, 912.3410; found 912.3455; HPLC (Method B) Rt=4.25.

N-(8-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)octyl)-6-(6-(5-((3aS,4R,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)hexanamide(123c)

121c (5.7 mg, 0.013 mmol), EZ-Link® NHS-LC_LC-Biotin (8.1 mg, 0.014mmol) and DIEA (3.4 mg, 4.5 μL, 0.026 mmol) in DMF (0.5 ml) was stirredat rt for 1 h. The reaction mixture was concentrated under reducedpressure and resulting residue was purified by preparatory TLC(CH₂Cl₂-MeOH—NH₃ (7N), 10:1) to give 3.7 mg (34%) of 123c. ¹H NMR (600MHz, CD₂Cl₂+5 drops of CD₃OD) δ 8.15 (s, 1H), 7.29 (t, J=1.8 Hz, 1H),7.26 (d, J=1.7 Hz, 2H), 4.40-4.42 (m, 1H), 4.20-4.23 (m, 1H), 4.12 (t,J=7.4 Hz, 2H), 3.03-3.10 (m, 8H), 2.79-2.85 (m, 1H), 2.58-2.64 (m, 1H),2.03-2.13 (m, 8H), 1.25-1.69 (m, 27H); HRMS (ESI) m/z [M+H]⁺ calcd. forC₄₁H₆₁Cl₂N₁₀O₄S₂, 891.3707; found 891.3696; HPLC (Method B) Rt=4.52.

5-(3-(3-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)thioureido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoicacid, WS-13-FITC2 (124a)

121a (9.4 mg, 0.0255 mmol), FITC (10.7 mg, 0.0281 mmol) and Et₃N (0.1mL) in DMF (0.5 mL) was stirred for 12 h at rt. The reaction mixture wasconcentrated under reduced pressure and the residue was purified by HPLCto give 14.8 mg (76%) of 124a. ¹H NMR (600 MHz, MeOH-d₄) δ 8.26 (s, 1H),8.17 (s, 1H), 7.72 (d, J=8.2 Hz, 1H), 7.54 (d, J=1.8 Hz, 2H), 7.43 (t,J=1.8 Hz, 1H), 7.11 (d, J=8.2 Hz, 1H), 6.83 (d, J=8.6 Hz, 2H), 6.76 (s,2H), 6.62 (d, J=8.7 Hz, 2H), 4.35 (t, J=6.8 Hz, 2H), 3.57 (m, 2H), 2.17(q, J=6.8 Hz, 2H); HRMS (ESI) m/z [M+H]⁺ calcd. for C₃₅H₂₆Cl₂N₇O₅S₂,758.0814; found 758.0818.

5-(3-(6-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)hexyl)thioureido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoicacid, WS-13-FITC3 (124b)

121b (7.2 mg, 0.0175 mmol), FITC (7.5 mg, 0.0193 mmol) and Et₃N (0.1 mL)in DMF (0.5 mL) was stirred for 12 h at rt. The reaction mixture wasconcentrated under reduced pressure and the residue was purified by HPLCto give 12.4 mg (86%) of 124b. ¹H NMR (600 MHz, MeOH-d₄) δ 8.26 (s, 1H),8.13 (s, 1H), 7.67 (d, J=7.7 Hz, 1H), 7.50 (d, J=1.9 Hz, 2H), 7.43 (t,J=1.8 Hz, 1H), 7.09 (d, J=8.2 Hz, 1H), 6.77 (d, J=8.7 Hz, 2H), 6.71 (s,2H), 6.56 (d, J=8.8 Hz, 2H), 4.25 (t, J=7.1 Hz, 2H), 3.88 (m, 2H), 1.76(q, J=7.0 Hz, 2H), 1.54 (q, J=6.9 Hz, 2H), 1.25-1.35 (m, 4H); HRMS (ESI)m/z [M+H]⁺ calcd. for C₃₅H₃₂Cl₂N₇O₅S₂, 800.1324; found 800.1329.

5-(3-(8-(6-Amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)octyl)thioureido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoicacid, WS-13-FITC4 (124c)

121c (6.4 mg, 0.0146 mmol), FITC (6.0 mg, 0.0153 mmol) and Et₃N (0.1 mL)in DMF (0.5 mL) was stirred for 12 h at rt. The reaction mixture wasconcentrated under reduced pressure and the residue was purified by HPLCto give 8.3 mg (72%) of 124c. ¹H NMR (600 MHz, MeOH-d₄) δ 8.30 (s, 1H),8.18 (s, 1H), 7.93 (d, J=8.2 Hz, 1H), 7.49 (d, J=1.7 Hz, 2H), 7.48 (t,J=1.7 Hz, 1H), 7.15 (d, J=8.2 Hz, 1H), 6.79-6.83 (m, 4H), 6.64 (d, J=8.7Hz, 2H), 4.27 (t, J=7.3 Hz, 2H), 3.62 (m, 2H), 1.82 (q, J=6.8 Hz, 2H),1.65 (q, J=6.9 Hz, 2H), 1.21-1.39 (m, 8H); HRMS (ESI) m/z [M+H]⁺ calcd.for C₄₀H3₆Cl₂N₇O₅S₂, 828.1596; found 828.1609.

6.6 Synthesis of [¹³¹I]-HJP-V-149 of Formula 125 (Scheme 23)

8-((3-chloro-5-(trimethylstannyl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine(124, HJP-VI-81)

A mixture of 53 (15 mg, 0.0298 mmol, 1 eq.), [(Me)₃Sn]₂ (4 eq.), LiCl (2eq.) and Pd(PPh₃)₄ (10-20 mol %) in dioxane (1 mL) in a 10 mL RBFequipped with a magnetic stir bar and rubber septum was evacuated andback filled with nitrogen. This was repeated four times then thereaction mixture was heated under nitrogen at 90° C. for 15 h. Solventwas removed under reduced pressure and the resulting residue waspurified by preparatory TLC (DCM:EtOAc:hexane:MeOH—NH₃ (7N) at 4:2:4:1,2×) to yield compound 124. Yield, 11.2 mg (70%). ¹H NMR (600 MHz, CDCl₃)δ 8.32 (s, 1H), 7.40-7.42 (m, 1H), 7.36-7.37 (m, 1H), 7.30-7.32 (m, 1H),5.84 (br s, 2H), 4.30 (t, J=6.8 Hz, 2H), 2.74-2.77 (m, 1H), 2.56 (t,J=6.7 Hz, 2H), 1.97-2.03 (m, 2H), 1.07 (d, J=6.2 Hz, 6H), 0.31 (s, 9H);¹³C NMR (150 MHz, CDCl₃) δ 154.5, 153.0, 151.6, 146.6, 145.3, 135.5,135.2, 135.1, 132.1, 130.0, 120.1, 48.9, 43.5, 41.5, 29.8, 22.5, −9.2.

Synthesis of [¹³¹I]-HJP-V-149 (125)

20 μg of Me₃Sn precursor 124 was dissolved in 25 μL methanol inEppendorf tube and to the resulting solution, [¹³¹I]—NaI solution wasadded (0.2 mCi in 2 μl in 0.1N NaOH) and the solution was vortex. Tothis solution 2 μl of chloramine-T (2 mg/ml acetic acid) was added andvortexed and allowed to react for 1 min and centrifuged at 300 rpm for15 s. Purification was achieved by passing through C-18 250×4.6 mm, RPLuna HPLC column (Phenomenex Torrance, Calif., C18, 5μ, 110° A), usingtwo solvent system of 0.1% TFA(A) and acetonitrile(B) as eluant under agradient of 20-80% B (from 3-10 min) with a flow rate of 1 ml/min. Theproduct has a retention time of about 9.7 minutes, under the conditionsdescribed above. HPLC profile of purified [¹³¹I]-Compound HJP-V-149(125)

6.7 Hsp90 Paralog Competition Assays

The Hsp90 FP competition assays were performed on an Analyst GTinstrument (Molecular Devices, Sunnyvale, Calif.) and carried out inblack 96-well microplates (Corning #3650) in a total volume of 100 μL ineach well. A stock of 10 μM Cy3B-GM and 115a was prepared in DMSO anddiluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT,5 mM MgCl2, 20 mM Na2MoO4, and 0.01% NP40 with 0.1 mg/mL BGG). To eachwell was added the fluorescent dye labeled Hsp90 ligand (6 nM Cy3B-GMfor Hsp90α, Hsp90β and Grp94 a.nd 3 nM of 115a for Trap-1), protein (10nM Hsp90α, 10 nM Hsp90β, 10 nM Grp94, 30 nM Trap-1) and tested inhibitor(initial stock in DMSO) in a final volume of 100 μL Felts buffer.Compounds were added in duplicate or triplicate wells. For each assay,background wells (buffer only), tracer controls (free, fluorescent dyelabeled Hsp90 ligand only) and bound controls (fluorescent dye labeledHsp90 ligand in the presence of protein) were included on each assayplate. The assay plate was incubated on a shaker at 4° C. for 24 h andthe FP values in mP were measured. The fraction of fluorescent dyelabeled Hsp90 ligand bound to Hsp90 was correlated to the mP value andplotted against values of competitor concentrations. The inhibitorconcentration at which 50% of bound fluorescent dye labeled Hsp90 ligandwas displaced was obtained by fitting the data. For cy3B-GM, anexcitation filter at 530 nm and an emission filter at 580 nm were usedwith a dichroic mirror of 561 nm. For 115a, an excitation filter at 485nm and an emission filter at 530 nm were used with a dichroic mirror of505 nm. All experimental data were analyzed using SOFTmax Pro 4.3.1 andplotted using Prism 4.0 (GraphPad Software Inc., San Diego, Calif.) andbinding affinity values are given as relative binding affinity values(EC50, concentration at which 50% of fluorescent ligand was competed offby compound).

The results of the competition assays for specific compounds produced inaccordance with the present disclosure are shown in Table 16 below:

TABLE 16 Com- MDA- pound Hsp90α Hsp90β Grp94 TRAP-1 MB- Structure code(μM) (μM) (μM) (μM) 468

PU-H6 >100 >100 0.35 ± 0.05

PU-H5 >>100 110 ± 20  6.2 ± 2.5

PU-H34 >>100 >>100 29 ± 9 

PU-H9 >>100 >>100 1.4 ± 0.2

PU-H47 >>100 >>100 100 ± 10 

PU-H46 >>100 >>100 >100

PU-H35 >>100 >>100 >100

PU-H48 >>100 >>100 106

PDP-I- 60-N9 2.18 ± 0.10 2.82 ± 0.14 0.53 ± 0.03 2.18 ± 0.55

PDP-I- 60-N3

PU-H1 >100 >100 1.13 ± 0.2

PU-H7 >100 ND 1.5 ± 0.2

PDP-I- 51-N9 >50 >10

PD-I- 51-N3 >50 >5

PU-H39 >300 >300 0.12 ± 0.04 145.4 ± 13.4 

WS-12 20.3 ± 1.2  0.41 ± 0.1 0.407

PDP-I- 54-N9  13.87 ± 1.3514 >50 0.66 ± 0.10 0.855 4.90 ± 0.27

PDP-I- 54-N3 >50 >10

PDP-I- 61-N9 >50 >50 0.28 ± 0.06 2.62 ± 0.28

PDP-I- 61-N3

PU-H3 >300 >300 1.63 ± 0.47 13.8 ± 1.8 

PU-H8 47.4 ± 2.2  22.4 ± 2.8  34.5 ± 3.5 

PU-H38 >500 >500 2.37 ± 0.79 9.6 ± 0.8

PU-H54 >250 >250 11.77 ± 3.24  54.1 ± 4.6 

PU-H44 >100 >100 46.76 >100

PDP- 122 >100 >100  0.5 ± 0.39 7.64 ± 2.15

HJP-V- 81 2.7 0.78 7.5

HJP-V- 82 31.3 0.23

HJP-V- 104 55.8 1.5

HJP-V- 105 >50 0.35

HJP-V- 83 33.0 2.9 8.8

HJP-V- 84 28.9 1.9 4.4

HJP-V- 85 9.2 2.3 2.6

HJP-V- 86 7.6 0.21

HJP-V- 88 1.2

HJP-V- 89 39.7 0.82

HJP-V- 91 18.9 1.4 0.63

HJP-V- 92 3.6 0.18

HJP-V- 93 >50 1.9

HJP-V- 116 >50 0.12-0.3 

HJP-V- 118 >50 1.03

HJP-V- 96 >50 1.9

HJP-V- 97 186.4 2.2

HJP-V- 98 >50 0.71

HJP-V- 100 97.9 0.32

HJP-V- 110 >50 0.60

HJP-V- 114 >50 2.48

HJP-V- 117 >50 0.92

HJP-V- 122

HJP-VI- 23 >50 1.5

HJP-VI- 25 >50

HJP-VI- 34 >50

HJP-VI- 36 3.4 0.21 5.85

HJP-VI- 32 14.2 >5

HJP-VI- 42 66.9 1.1

HJP-VI- 43 1.5 0.45 6.58

HJP-VI- 49 29.6 1.3 3.12

PU- H42 60.4 ± 1.6  >100 0.53 ± 0.10 5.1 ± 4.2

PU- H27 >300 >300 1.14 ± 0.14 203.7 ± 11.0 

PU- H2 100 ± 7   6.5 ± 2.3 0.5 ± 0.2

PU- H43 >>100 >>100 35.3 ± 5  

PU- H29 >>100 >>100 185 ± 15 

PU- H53 >>100 >>100 200

PU- H63 5.9 ± 0.5 2.5 ± 0.2 2.3 ± 0.1

PU- H4 >300 >300 11.45 ± 0.92  157.9 ± 9.7  

PU- H41 >>100 100 ± 11  100 ± 8  

PU- H45 >>100 >>100 >>100

WS- 14 8.56 2.43

WS- 13 27.3 ± 3.5  41.8 ± 1.3  0.22 ± 0.08 7.3

PDP-I- 13-N9 >50 >100 0.57 ± 0.08 10.09 ± 1.13 

PDP-I- 13-N3 >50 >100 12.78 ± 0.86  >100

PDP-I- 14-N9 >100 >100 0.27 ± 0.02 >100

PDP-I- 14-N3 >50 >5

PDP-I- 15-N9 >50 >50 0.46 ± 0.04 >50

PDP-I- 15-N3 >50 >10

PDP-I- 83-N9

PDP-I- 83-N3

PDP- 93A >50 >100 0.45 ± 0.03 4.47 ± 0.31

PDP- 93B >100 >100 >100 >100

PDP- 101B >100 >100 1.65 ± 0.31 >100

PDP- 101A >100 >100 >100 >100

PDP- 102A >100 >100 >5 >50

PDP- 102B >100 >100 10.27 ± 0.82  >25

PDP- 107A >100 >100 >5 >50

PDP- 107B >100 >100 11.60 >100

PDP- 109A >100 >100 1.11 ± 0.28 >100

PDP- 109B >100 >100 >5 >100

PDP- 110A >100 >100 4.34 ± 0.75 >100

PDP- 110B NA NA NA NA

PDP- 112A >100 >100 0.65 ± 0.05 >100

PDP- 112B >100 >100 >10 >100

PDP- 99A >100 >100 1.52 ± 0.03 >100

PDP- 99B >100 >100 >10 >100

PDP- 132 >100 >100 0.72 ± 0.02 44.38 ± 4.61 

HJP-V- 132 26.6 0.81

HJP-V- 123

HJP-V- 130 51.7 0.37

SO-III- 35A 87.6 0.776

SO-III- 36A 65.8 0.335

SO-III- 37A 26.1 0.234

SO-III- 39A 52.2 0.373

SO-III- 40A 97.2 0.768

SO-III- 75A 39.92 0.89

SO-III- 116A 47.7 0.44

SO-III- 128B 35.6 0.276 0.84

SO-III- 127B 40.4 0.345 0.83

SO-III- 103A >10 0.203

PDP-127 >100 >100 >2.5 >100

HJP-III- 26 60.29 >3 (7.73)

PDP-I- 59-N9 >100 >100 0.26 ± 0.07 >100

PDP-I- 59-N3

PDP-I- 53-N9 >100 >100 0.22 ± 0.02 >100

PDP-I- 53-N3

PDP- 108A >50 >100 0.133 >50

PDP- 120A >100 >100 1.207 1.873

PDP- 120B >100 >100 >10 >100

HJP-VI- 12 20 0.21 0.063

HJP-VI- 14 >50 0.16

HJP-VI- 18 152.03 0.9 0.122

HJP-VI- 50 >50 2.0

HJP-VI- 51 >50 1.2

HJP-VI- 52 >50 0.18

HJP-VI- 53 >50 0.63

HJP-VI- 58 >50 5.8

HJP-VI- 59 >50 7.4

HJP-VI- 62 >50 0.28

HJP-VI- 63 >50 0.34 0.226

HJP-VI- 64 >50 0.24

HJP-VI- 70 13.77 0.153 2.16

HJP-VI- 72 14.23 0.102

HJP-VI- 78 18.6 0.214 0.58

HJP-VI- 79 0.124

HJP-VI- 31 48.9 0.63 0.129

HJP-V- 147 72.5 0.86

HJP-V- 149 43.2 0.24 0.125

HJP-VI- 69 10 0.135 0.030

HJP-VI- 84

HJP-VI- 85 >50 0.186 0.164

HJP-VI- 86 20.94 0.156 0.053

HJP-VI- 4 41.8 0.29 0.212

HJP-VI- 5 >25 0.49 0.562

HJP-VI- 6 27.8 0.54 0.314

HJP-VI- 3 >25 2.7

HJP-VI- 7 47.5 0.26

HJP-VI- 8 47.3 0.58

HJP-VI- 9 13.8 0.26

HJP-VI- 10 7.9 0.16

HJP-VI- 28 0.22 0.286

HJP-VI- 29 25.9 1.2 0.516

HJP-VI- 30 16.7 0.32

HJP-VI- 38 25.9 0.75

HJP-VI- 39 21.3 0.28 0.166

HJP-VI- 44 12.3 0.83 0.917

HJP-VI- 45

HJP-VI- 46 15.9 1.9

HJP-VI- 47 >50 3.3

HJP-III- 29 4.51 2.41

HJP-III- 32 14.32 4.28

HJP-III- 33 7.60 3.45

PDP-I- 25-N9 4.82 ± 1.05 10.22 ± 1.62 0.42 ± 0.04 2.35 ± 0.09

PDP-I- 25-N3 >50 11.11

PU-H36 >250 >250 2.10 ± 0.56 65.5 ± 1.3 

PU-H37 >>100 >>100 20 ± 5

PU-H51 >200 >200 0.17 ± 0.11 78.4 ± 8.9 

PU-H52 >>100 >>100 2.08 ± 1.49 19.5 ± 0.2 

HJP-V- 103-N9 5.8 0.35

PDP-121 >100 >100 1.66 ± 0.48 2.20 ± 0.67

>100 >100 0.19 ± 0.01 6.04 ± 0.55

PDP- 125B >100 >100 5.208 58.824

PDP- 126A 15.72 ± 0.35  18.26 ± 1.29  0.27 ± 0.14 4.03 ± 0.89

PDP- 126B >100 >100 8.70 34.10

HJP-V- 145 0.68

PDP-I- 55-N9 >50 >5

PDP-I- 55-N3

PDP-I- 58-N9 >50 >10

PDP-I- 58-N3 >50 >10

PDP-I- 16-N3 >100 >5

PDP-I- 16-N9 >100 >100 2.16 ± 0.21 >100

PDP-I- 77-N9 >50 >2

PDP-I- 77-N3 >50 >5

PDP-I- 79-N9 >100 >100 2.78 ± 0.21 >100

PDP-I- 79-N3 >50 >5

PU1 110 ± 20 92 ± 2 3.2 ± 0.4

HJP-V- 36 >10 >5

HJP-V- 37 >10 0.7

HJP-V- 37T >25 0.13-0.17

HJP-V- 62M 24.1 5.4

HJP-V- 62T >25 4.7

HJP-V- 45 >10 0.5

HJP-V- 38 14.4 13.8

HJP-V- 39 >10 0.5

HJP-V- 39T >10 5.0

HJP-V- 54 >10 0.75

HJP-V- 55 >10 2.0

PU-27 >500 >250 59.30 ± 2.69  >300

PU-34 >500 >250 91.33 ± 2.16  227.2 ± 6.6  

SO-III- 154 >50 2.59

HJP-VI- 101 9.2 >5

SO-IV- 03 >50 >5

PDP- 117A >100 >100 1.58 34.42

PDP- 119A >100 >100 1.96 >100

HJP-V- 54T

HJP-V- 90

WS-11 >100 25

WS-13- N3 >100 >100 2.43 >100

HJP-V- 125

HJP-V- 140 >50 7.9

HJP-V- 134 >10 0.38

HJP-V1- 66 >5 1.9

1-32. (canceled)
 33. A compound of the Formula (III):

or a pharmaceutically acceptable salt thereof, wherein: (a) Y is—C(R^(Y))₂—, —S—, —NR—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—; (c) Z² is —N— or—CR¹⁰—, wherein R¹⁰ is H or unsubstituted or substituted—(C₁-C₆)aliphatic; (d) each of Z⁶, Z⁷ and Z⁸ are independently —C— or—N—, with the proviso that at least one of Z⁶—Z⁸ is —C—; (e) X¹ is —H,-halo, —N(R)₂, —OR, —CN, or unsubstituted or substituted—(C₁-C₆)aliphatic; (f) each of X⁴, X⁵, and X⁶ are independently —H,-halo, —SR, —N(R)₂, —OR, —CN, —NO₂, —CN, —C(O)R, —C(O)₂R, —S(O)R,—S(O)₂R, —C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R, —N(R)SO₂R,—OC(O)N(R)₂, unsubstituted or substituted —(C₁-C₆)aliphatic, or anunsubstituted or substituted group selected from (5- or 6-membered)aryl,(5- or 6-membered)arylalkyl, and (5- or 6-membered)heterocyclic aromaticor heterocyclic non-aromatic group; with the provisos that X⁴ is absentwhen Z⁶ is a nitrogen, X⁵ is absent when Z⁷ is a nitrogen and X⁶ isabsent when Z⁸ is a nitrogen; (g) R⁷ is—(C₁-C₆)aliphatic-N⁺—(R²)(R³)(R⁴), —(C₁-C₆)aliphatic-N—R³R⁴,—(C₁-C₆)aliphatic-C(═O)N—R³R⁴, —(C₁-C₆)aliphatic-R³R⁴,—(C₁-C₆)aliphatic-R²R³R⁴, —(C₁-C₆)aliphatic-N—CR²R³R⁴,—(C₁-C₆)aliphatic-C(halo)₃, —(C₁-C₆)aliphatic-alkenyl,—(C₁-C₆)aliphatic-alkynyl, —(C₁-C₆)aliphatic-(C₃-C₈)cycloalkyl,—(C₁-C₆)aliphatic-(C₃-C₆)heterocycloalkyl, —(C₁-C₆)aliphatic-phenyl,—(C₁-C₆)aliphatic-(5 or 6-membered)heteroaryl, —(C₁-C₆)aliphatic-cyano,with the proviso that when all of R²-R⁴ are present the compound furthercomprises a pharmaceutically acceptable counter ion; (h) Q is fusedbenzo, fused (5- or 6-membered)heteroaryl, a fused 4 to 7-memberedcyloalkyl ring or a fused 4- to 7-membered non-aromatic heterocyclicring; (i) R² and R³ are independently hydrogen, —N(R)₂, —CH₂CH(OH)R⁴,—CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴, or unsubstituted or substituted—(C₁-C₆)aliphatic, or R³ and R⁴ form an unsubstituted or substituted 3-to 7-membered heterocyclic ring when taken together with the nitrogen towhich they are attached; (j) R⁴ is hydrogen, halogen, or unsubstitutedor substituted —(C₁-C₆)aliphatic; (k) each R⁸ is independently —H,-halo, —N(R)₂, —OR, —CN, or a unsubstituted or substituted selected from—CH₂-phenyl or —(C₁-C₆)aliphatic; (l) each R^(Y) is independently R,—OR, or halo; (m) a is an integer selected from 0, 1 and 2; and (n) eachR is independently hydrogen, unsubstituted C₁₋₆ aliphatic, or C₁₋₆aliphatic substituted with halo, —OH, —CN, or —NH₂; wherein eachsubstituted group is substituted with one or more groups selected fromhalo, —N(R)₂, —OR, —CN, oxo, unsubstituted C₁₋₆ aliphatic, or C₁₋₆aliphatic substituted with halo, —OH, —CN, or —NH₂.
 34. The compound ofclaim 33 or a pharmaceutically acceptable salt thereof, wherein: (a) Yis —CH₂—, —S—, —N—, —O—,

(b) each of Z¹ and Z³ are independently —C— or —N—; (c) Z² is —CH—, —N—,or —CR¹⁰—, wherein R¹⁰ is —(C₁-C₆)alkyl; (d) each of Z⁶, Z⁷ and Z⁸ areindependently —C— or —N—, with the proviso that at least one of Z⁶—Z⁸ is—C—; (e) X¹ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl,—CH₂OH, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, or—OCH₂(halo); (f) each of X⁴, X⁵, and X⁶ are independently —H, -halo,—NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, —OCH₂(halo), or a (5- or6-membered)aryl, heterocyclic aromatic, or non-aromatic group selectedfrom pyridyl, furyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl,thiazolidinyl, thiadiazolyl, thiazolyl, isoxazolyl, pyrazolyl,isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl,2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl,with the provisos that X⁴ is absent when Z⁶ is a nitrogen, X⁵ is absentwhen Z⁷ is a nitrogen and X⁶ is absent when Z⁸ is a nitrogen; (g) R⁷ is—(CH₂)_(m)—N⁺—(R²)(R³)(R⁴), —(CH₂)_(m)—N—R³R⁴, —(CH₂)_(m)—C(═O)N—R³R⁴,—(CH₂)_(m)R³R⁴, —(CH₂)_(m)—C(halo)₃, —(CH₂)_(m)-alkenyl,(CH₂)_(m)-alkenyl-CH₃, —(CH₂)_(m)-alkynyl, (CH₂)_(m)-alkynyl-CH₃,(CH₂)_(m)—(C₃-C₈)cycloalkyl, —(CH₂)_(m)—(C₃-C₈)heterocycloalkyl,—(CH₂)_(m)-phenyl, —(CH₂)_(m)-(5 or 6-membered)heteroaryl,—(CH₂)_(m)-cyano, where m is 1, 2, 3, 4 or 5 and where the cyloalkyl,heterocycle or phenyl is unsubstituted or substituted with one or moreX¹ groups, with the proviso that when all of R²-R⁴ are present thecompound further comprises a pharmaceutically acceptable counter ion;(h) Q is fused benzo, fused (5- or 6-membered)heteroaryl, a fused 4 to7-membered cyloalkyl ring or a fused 4- to 7-membered non-aromaticheterocyclic ring selected from pyrrolo, pyridino, pyrimidino, pyrazino,pyridazino, oxadiazolo, thiadiazolo, dioxolano, imidazolo, orimidazo[1,2-a]pyridine; (i) R² and R³ are independently hydrogen,methyl, ethyl, ethenyl, ethynyl, propyl, butyl, pentyl, hexyl,isopropyl, t-butyl, isobutyl, —C(halo)₃, —CH(halo)₂, —CH₂(halo),—CH₂C(halo)₃, —CHCH(halo)₂, CHCH₂(halo), —CH₂OH, —CH₂CH₂OH,—CH₂C(CH₃)₂OH, —CH₂CH(CH₃)OH, —C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH,—CH(CH₃)CH(OH)R⁴, —CH₂CH(OH)R⁴, —CH₂SO₂NHR⁴, —CH₂NHSO₂R⁴, or R² and R³form an unsubstituted or substituted aziridine, azetidine, pyrrolidineor piperidine ring when taken together with the nitrogen to which theyare attached; (j) R⁴ is hydrogen, methyl, ethyl, isopropyl, t-butyl,isobutyl, or —C(halo)₃; (k) R⁸ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl,—O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃,—OCH(halo)₂, and —OCH₂(halo), and X³, X⁴, X⁵, and X⁶ are independentlyselected from —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl,—CH₂OH, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂,—OCH₂(halo), pyridyl, furyl, phenyl, benzyl, thiophenyl, pyrrolyl,oxazolyl, imidazolyl, thiazolidinyl, thiadiazolyl, thiazolyl,isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,triazinyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, or terrahydrothiopyranyl;and (l) a is an integer selected from 0, 1 and
 2. 35. A compound of theFormula (IV):

or a pharmaceutically acceptable salt thereof, wherein: (a) Y is—C(R^(Y))₂—, —S—, —NR—, —O—,

(b) each of Z¹, Z³, Z⁹, Z¹⁰, Z¹¹, and Z¹² are independently —CH— or —N—;(c) Z² is —N— or —CR¹⁰—, wherein R¹⁰ is H or unsubstituted orsubstituted —(C₁-C₆)aliphatic; (d) each of X⁸ and X⁹ are independently—CH—, —S—, —N—, or —O—; (e) X¹ is —H, -halo, —N(R)₂, —OR, —CN, orunsubstituted or substituted —(C₁-C₆)aliphatic; (f) R⁷ is—(C₁-C₆)aliphatic-N⁺—(R²)(R³)(R⁴), —(C₁-C₆)aliphatic-N—R³R⁴,—(C₁-C₆)aliphatic-C(═O)N—R³R⁴, —(C₁-C₆)aliphatic-R³R⁴,—(C₁-C₆)aliphatic-R²R³R⁴, —(C₁-C₆)aliphatic-N—CR²R³R⁴,—(C₁-C₆)aliphatic-C(halo)₃, —(C₁-C₆)aliphatic-alkenyl,—(C₁-C₆)aliphatic-alkynyl, —(C₁-C₆)aliphatic-(C₃-C₆)cycloalkyl,—(C₁-C₆)aliphatic-(C₃-C₆)heterocycloalkyl, —(C₁-C₆)aliphatic-phenyl,—(C₁-C₆)aliphatic-(5 or 6-membered)heteroaryl, —(C₁-C₆)aliphatic-cyano,with the proviso that when all of R²-R⁴ are present the compound furthercomprises a pharmaceutically acceptable counter ion; (g) R² and R³ areindependently hydrogen, —N(R)₂, —CH₂CH(OH)R⁴, —CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴,—CH₂NHSO₂R⁴, or unsubstituted or substituted —(C₁-C₆)aliphatic, or R³and R⁴ form an unsubstituted or substituted 3- to 7-memberedheterocyclic ring when taken together with the nitrogen to which theyare attached; (h) R⁴ is hydrogen, halogen, or unsubstituted orsubstituted —(C₁-C₆)aliphatic; (i) each R⁸ is independently —H, -halo,—N(R)₂, —OR, —CN, or unsubstituted or substituted —(C₁-C₆)aliphatic; (j)R⁹ is —H, (C₁-C₆)aliphatic-cycloalkyl,—(C₁-C₆)aliphatic-heterocycloalkyl, —(C₁-C₆)aliphatic-aryl,—(C₁-C₆)aliphatic-heteroaryl, or —(C₁-C₆)aliphatic-cyano, wherein eachcycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted orsubstituted, with the proviso that R⁹ is absent when X⁹ is —S— or —O—;(k) each R^(Y) is independently R, —OR, or halo; (l) a is an integerselected from 0, 1 and 2; and (m) each R is independently hydrogen,unsubstituted C₁₋₆ aliphatic, or C₁₋₆ aliphatic substituted with halo,—OH, —CN, or —NH₂; and wherein each substituted group is substitutedwith one or more groups selected from halo, —N(R)₂, —OR, —CN, oxo,unsubstituted C₁₋₆ aliphatic, or C₁₋₆ aliphatic substituted with halo,—OH, —CN, or —NH₂.
 36. The compound of claim 35 or a pharmaceuticallyacceptable salt thereof, wherein: (a) Y is —CH₂—, —S—, —N—, —O—,

(b) each of Z¹, Z³, Z⁹, Z¹⁰, Z¹¹ and Z¹² are independently —CH— or —N—;(c) Z² is —CH—, —N—, or —CR¹⁰—, wherein R¹⁰ is —(C₁-C₆)alkyl; (d) eachof X⁸ and X⁹ are independently —CH₂—, —S—, —N—, or —O—; (e) X¹ is —H,-halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃,—CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂, or —OCH₂(halo); (f) R⁷is —(CH₂)_(m)—N⁺—(R²)(R³)(R⁴), —(CH₂)_(m)—N—R³R⁴,—(CH₂)_(m)—C(═O)N—R³R⁴, —(CH₂)_(m)—R³R⁴, —(CH₂)_(m)—C(halo)₃,—(CH₂)_(m)-alkenyl, (CH₂)_(m)-alkenyl-CH₃, —(CH₂)_(m)-alkynyl,(CH₂)_(m)-alkynyl-CH₃, (CH₂)_(m)—(C₃-C₈)cycloalkyl,—(CH₂)_(m)—(C₃-C₈)heterocycloalkyl, —(CH₂)_(m)-phenyl, —(CH₂)_(m)-(5 or6-membered)heteroaryl, —(CH₂)_(m)-cyano, where m is 1, 2, 3, 4 or 5 andwhere the cyloalkyl, heterocycle or phenyl is unsubstituted orsubstituted with one or more X¹ groups, with the proviso that when allof R²-R⁴ are present the compound further comprises a pharmaceuticallyacceptable counter ion; (g) R² and R³ are independently hydrogen,methyl, ethyl, ethenyl, ethynyl, propyl, butyl, pentyl, hexyl,isopropyl, t-butyl, isobutyl, —C(halo)₃, —CH(halo)₂, —CH₂(halo),—CH₂C(halo)₃, —CHCH(halo)₂, CHCH₂(halo), —CH₂OH, —CH₂CH₂OH,—CH₂C(CH₃)₂OH, —CH₂CH(CH₃)OH, —C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH or R² and R³form an unsubstituted or substituted aziridine, azetidine, pyrrolidineor piperidine ring when taken together with the nitrogen to which theyare attached; (h) R⁴ is hydrogen, methyl, ethyl, isopropyl, t-butyl,isobutyl, or —C(halo)₃; (i) R⁸ is —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl,—O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃,—OCH(halo)₂, and —OCH₂(halo), and X³, X⁴, X⁵, and X⁶ are independentlyselected from —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl,—CH₂OH, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃, —OCH(halo)₂,—OCH₂(halo), pyridyl, furyl, phenyl, benzyl, thiophenyl, pyrrolyl,oxazolyl, imidazolyl, thiazolidinyl, thiadiazolyl, thiazolyl,isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,triazinyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, or terrahydrothiopyranyl;(j) R⁹ is —H, (CH₂)n-cycloalkyl, —(CH₂)n-heterocycloalkyl, —(CH₂)n-aryl,—(CH₂)n-heteroaryl, or —(CH₂)-cyano, wherein said cycloalkyl,heterocycloalkyl, aryl or heteroaryl is optionally substituted with oneor more X¹ groups; (k) a is an integer selected from 0, 1 and 2; and (l)n is an integer selected from 1, 2, 3 or
 4. 37. A compound of theFormula (V):

or a pharmaceutically acceptable salt thereof, wherein: (a) Y is—C(R^(Y))₂—, —S—, —NR—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—; (c) Z² is —N— or—CR¹⁰—, wherein R¹⁰ is H or unsubstituted or substituted—(C₁-C₆)aliphatic; (d) each of Z⁴, Z⁵, Z⁶, Z⁷ and Z⁸ are independently—C— or —N—, with the proviso that no three consecutive Z⁴ through Z⁸ areN; (e) X¹ is —H, -halo, —N(R)₂, —OR, —CN, or unsubstituted orsubstituted —(C₁-C₆)aliphatic; (f) each of X⁴, X⁵, and X⁶ areindependently —H, -halo, —SR, —N(R)₂, —OR, —CN, —NO₂, —CN, —C(O)R,—C(O)₂R, —S(O)R, —S(O)₂R, —C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R, —N(R)C(O)R,—N(R)SO₂R, —OC(O)N(R)₂, unsubstituted or substituted —(C₁-C₆)aliphatic,or an unsubstituted or substituted group selected from (5- or6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or6-membered)heterocyclic aromatic or heterocyclic non-aromatic group;with the provisos that at least one of X², X⁴ and X⁵ is —H and that X²is absent when Z⁴ is —N—, X³ is absent when Z⁵ is —N—, X⁴ is absent whenZ⁶ is —N— and X⁵ is absent when Z⁷ is —N—; (g) each of X² and X³ areindependently selected from (1) —H, -halo, —SR, —N(R)₂, —OR, —CN, —NO₂,—CN, —C(O)R, —C(O)₂R, —S(O)R, —S(O)₂R, —C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R,—N(R)C(O)R, —N(R)SO₂R, —OC(O)N(R)₂, unsubstituted or substituted—(C₁-C₆)aliphatic, or an unsubstituted or substituted group selectedfrom (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or6-membered)heterocyclic aromatic or heterocyclic non-aromatic group; or(2) X² and X³ taken together form a fused benzo or fused (5- or6-membered) heteroaryl that may be substituted with one or more R⁸groups; (h) R⁷ is —(C₁-C₆)aliphatic-N⁺—(R²)(R³)(R⁴),—(C₁-C₆)aliphatic-N—R³R⁴, —(C₁-C₆)aliphatic-C(═O)N—R³R⁴,—(C₁-C₆)aliphatic-R³R⁴, —(C₁-C₆)aliphatic-R²R³R⁴,—(C₁-C₆)aliphatic-N—CR²R³R⁴, —(C₁-C₆)aliphatic-C(halo)₃,—(C₁-C₆)aliphatic-alkenyl, —(C₁-C₆)aliphatic-alkynyl,—(C₁-C₆)aliphatic-(C₃-C₈)cycloalkyl,—(C₁-C₆)aliphatic-(C₃-C₈)heterocycloalkyl, —(C₁-C₆)aliphatic-phenyl,—(C₁-C₆)aliphatic-(5 or 6-membered)heteroaryl, —(C₁-C₆)aliphatic-cyano,with the proviso that when all of R²-R⁴ are present the compound furthercomprises a pharmaceutically acceptable counter ion; (i) R² and R³ areindependently hydrogen, —N(R)₂, —CH₂CH(OH)R⁴, —CH(OH)CH₂R⁴, —CH₂SO₂NHR⁴,—CH₂NHSO₂R⁴, or unsubstituted or substituted —(C₁-C₆)aliphatic, or R³and R⁴ form an unsubstituted or substituted 3- to 7-memberedheterocyclic ring when taken together with the nitrogen to which theyare attached; (j) R⁸ is —H, -halo, —SR, —N(R)₂, —OR, —CN, —NO₂, —CN,—C(O)R, —C(O)₂R, —S(O)R, —S(O)₂R, —C(O)N(R)₂, —SO₂N(R)₂, —OC(O)R,—N(R)C(O)R, —N(R)SO₂R, —OC(O)N(R)₂, unsubstituted or substituted—(C₁-C₆)aliphatic, or an unsubstituted or substituted group selectedfrom (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or6-membered)heterocyclic aromatic or heterocyclic non-aromatic group; (k)R⁴ is hydrogen, halogen, or unsubstituted or substituted—(C₁-C₆)aliphatic; and (l) each R is independently hydrogen,unsubstituted C₁₋₆ aliphatic, or C₁₋₆ aliphatic substituted with halo,—OH, —CN, or —NH₂; wherein each substituted group is substituted withone or more groups selected from halo, —N(R)₂, —OR, —CN, oxo,unsubstituted C₁₋₆ aliphatic, or C₁₋₆ aliphatic substituted with halo,—OH, —CN, or —NH₂.
 38. The compound of claim 37 or a pharmaceuticallyacceptable salt thereof, wherein: (a) Y is —CH₂—, —S—, —N—, —O—,

(b) each of Z¹ and Z³ are independently —CH— or —N—; (c) Z² is —CH—,—N—, or —CR¹⁰—, wherein R¹⁰ is —(C₁-C₆)alkyl; (d) each of Z⁴, Z⁵, Z⁶, Z⁷and Z⁸ are independently —CH— or —N—, with the proviso that no threeconsecutive Z⁴ through Z⁸ are N; (e) X¹ is —H, -halo, —NH₂, —CN,—(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, or —OCH₂(halo); (f) each of X⁴, X⁵,and X⁶ are independently —H, -halo, —NH₂, —CN, —(C₁-C₆)alkyl,—O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂, —CH₂(halo), —OC(halo)₃,—OCH(halo)₂, —OCH₂(halo), pyridyl, furyl, thiophenyl, pyrrolyl,oxazolyl, imidazolyl, thiazolidinyl, thiadiazolyl, thiazolyl,isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,triazinyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl(g) each of X² and X³ are independently selected from (1) —H, -halo,—NH₂, —CN, —(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, —OCH₂(halo), pyridyl, furyl,thiophenyl, pyrrolyl, oxazolyl, imidazolyl, thiazolidinyl, thiadiazolyl,thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl,pyrimidinyl, triazinyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl,tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, ortetrahydrothiopyranyl; and (2) X² and X³ taken together form a fusedbenzo or fused (5- or 6-membered) heteroaryl that may be substitutedwith one or more R⁸ groups; (h) R⁷ is —(CH₂)_(m)—N⁺—(R²)(R³)(R⁴),—(CH₂)_(m)—N—R³R⁴, —(CH₂)_(m)—C(═O)N—R³R⁴, —(CH₂)_(m)—C(halo)₃,—(CH₂)_(m)-alkenyl, (CH₂)_(m)-alkenyl-CH₃, —(CH₂)_(m)-alkynyl,(CH₂)_(m)-alkynyl-CH₃, (CH₂)_(m)—(C₃-C₈)cycloalkyl,—(CH₂)_(m)—(C₃-C₈)heterocycloalkyl, —(CH₂)_(m)-phenyl, —(CH₂)_(m)-(5 or6-membered)heteroaryl, —(CH₂)_(m)-cyano, where m is 1, 2, 3, 4 or 5 andwhere the cyloalkyl, heterocycle or phenyl is unsubstituted orsubstituted with one or more X¹ groups, with the proviso that when allof R²-R⁴ are present the compound further comprises a pharmaceuticallyacceptable counter ion; (i) R² and R³ are independently hydrogen,methyl, ethyl, ethenyl, ethynyl, propyl, butyl, pentyl, hexyl,isopropyl, t-butyl, isobutyl, —C(halo)₃, —CH(halo)₂, —CH₂(halo),—CH₂C(halo)₃, —CHCH(halo)₂, CHCH₂(halo), —CH₂OH, —CH₂CH₂OH,—CH₂C(CH₃)₂OH, —CH₂CH(CH₃)OH, —C(CH₃)₂CH₂OH, —CH(CH₃)CH₂OH or R² and R³form an unsubstituted or substituted aziridine, azetidine, pyrrolidineor piperidine ring when taken together with the nitrogen to which theyare attached; (j) R⁴ is hydrogen, methyl, ethyl, isopropyl, t-butyl,isobutyl, or —C(halo)₃; and (k) R⁸ is —H, -halo, —NH₂, —CN,—(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, and —OCH₂(halo), and X³, X⁴, X⁵,and X⁶ are independently selected from —H, -halo, —NH₂, —CN,—(C₁-C₆)alkyl, —O(C₁-C₆)alkyl, —CH₂OH, —C(halo)₃, —CH(halo)₂,—CH₂(halo), —OC(halo)₃, —OCH(halo)₂, —OCH₂(halo), pyridyl, furyl,phenyl, benzyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl,thiazolidinyl, thiadiazolyl, thiazolyl, isoxazolyl, pyrazolyl,isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl, morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl,2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, or terrahydrothiopyranyl.39. The compound of claim 37 or 38, having the following formula:

or a pharmaceutically acceptable salt thereof.
 40. The compound of claim37 or 38, having the following formula:

or a pharmaceutically acceptable salt thereof.
 41. The compound of claim37 or 38, having the following formula:

or a pharmaceutically acceptable salt thereof.
 42. The compound of claim37 or 38, having the following formula:

or a pharmaceutically acceptable salt thereof.
 43. The compound of claim37 or 38, having the following formula:

or a pharmaceutically acceptable salt thereof.
 44. The compound of claim37 or 38, having the following formula:

or a pharmaceutically acceptable salt thereof.
 45. The compound of claim37 or 38, having the following formula:

or a pharmaceutically acceptable salt thereof. 46-47. (canceled)
 48. Acompound having the following formula:

pharmaceutically acceptable salt thereof. 49-50. (canceled)
 51. Apharmaceutical composition comprising a compound of claim 33, and apharmaceutically acceptable excipient.
 52. (canceled)
 53. A method oftreating cancer, autoimmune diseases, inflammatory diseases,neurodegenerative diseases, rheumatoid arthritis, or diabetes in apatient, comprising administering a therapeutically effective amount ofa compound of claim 33, or a pharmaceutically acceptable salt thereof.54-78. (canceled)
 79. A pharmaceutical composition comprising a compoundof claim 35, and a pharmaceutically acceptable excipient.
 80. Apharmaceutical composition comprising a compound of claim 37, and apharmaceutically acceptable excipient.
 81. A pharmaceutical compositioncomprising a compound of claim 48, and a pharmaceutically acceptableexcipient.
 82. A method of treating cancer, autoimmune diseases,inflammatory diseases, neurodegenerative diseases, rheumatoid arthritis,or diabetes in a patient, comprising administering a therapeuticallyeffective amount of a compound of claim 35, or a pharmaceuticallyacceptable salt thereof.
 83. A method of treating cancer, autoimmunediseases, inflammatory diseases, neurodegenerative diseases, rheumatoidarthritis, or diabetes in a patient, comprising administering atherapeutically effective amount of a compound of claim 37, or apharmaceutically acceptable salt thereof.
 84. A method of treatingcancer, autoimmune diseases, inflammatory diseases, neurodegenerativediseases, rheumatoid arthritis, or diabetes in a patient, comprisingadministering a therapeutically effective amount of a compound of claim48, or a pharmaceutically acceptable salt thereof.